0 High-affinity binding of phosphatidylinositol, otherFeat=[]-->, belongsTo=title 1 4-phosphate by Legionella pneumophila DrrA, otherFeat=[]-->, belongsTo=title 2 Stefan Schoebel, Wulf Blankenfeldt, Roger S. Goody+ &Aymelt Itzen++, otherFeat=[]-->, belongsTo=title 3 Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, North Rhine-Westphalia, Germany, otherFeat=[]-->, belongsTo=title 4 The DrrA protein of Legionella pneumophila is involved, otherFeat=[]-->, belongsTo=parr 5 in mistargeting of endoplasmic reticulum-derived vesicles to, otherFeat=[]-->, belongsTo=parr 6 Legionella-containing vacuoles through recruitment of the, otherFeat=[]-->, belongsTo=parr 7 small GTPase Rab1. To this effect, DrrA binds specifically to, otherFeat=[]-->, belongsTo=parr 8 phosphatidylinositol 4-phosphate (PtdIns(4)P) lipids on the, otherFeat=[]-->, belongsTo=parr 9 cytosolic surface of the phagosomal membrane shortly after, otherFeat=[]-->, belongsTo=parr 10 infection. In this study, we present the atomic structure of the, otherFeat=[]-->, belongsTo=parr 11 PtdIns(4)P-binding domain of a protein (DrrA) from a human, otherFeat=[]-->, belongsTo=parr 12 pathogen. A detailed kinetic investigation of its interaction with, otherFeat=[]-->, belongsTo=parr 13 PtdIns(4)P reveals that DrrA binds to this phospholipid with, as, otherFeat=[]-->, belongsTo=parr 14 yet unprecedented, high affinity, suggesting that DrrA can sense, otherFeat=[]-->, belongsTo=parr 15 a very low abundance of the lipid., otherFeat=[]-->, belongsTo=parr 16 Keywords: DrrA; Legionella; phosphatidylinositol 4-phosphate;, otherFeat=[]-->, belongsTo=parr 17 Rab1; SidM, otherFeat=[]-->, belongsTo=parr 18 EMBO reports (2010) 11, 598?604. doi:10.1038/embor.2010.97, otherFeat=[]-->, belongsTo=parrnote 19 INTRODUCTION, otherFeat=['U']-->, belongsTo=title 20 The pathogen Legionella pneumophila causes Legionnaires', otherFeat=[]-->, belongsTo=parr 21 disease by infecting human lung macrophages (Muder et al,, otherFeat=[]-->, belongsTo=parr 22 1986). Shortly after uptake, the bacterium establishes the, otherFeat=[]-->, belongsTo=parr 23 Legionella-containing vacuole (LCV) as an intracellular replicative, otherFeat=[]-->, belongsTo=parr 24 organelle, from which it injects several proteins through its, otherFeat=[]-->, belongsTo=parr 25 type IV secretion system into the cytosol of the host cell (Isberg, otherFeat=[]-->, belongsTo=parr 26 et al, 2009). One of the substrates of the type IV secretion system, otherFeat=[]-->, belongsTo=parr 27 is DrrA, also known as SidM, a 647-amino-acid protein that is, otherFeat=[]-->, belongsTo=parr 28 involved in redirecting endoplasmic reticulum (ER)-derived, otherFeat=[]-->, belongsTo=parr 29 vesicles to the LCV. DrrA consists of three domains, the central, otherFeat=[]-->, belongsTo=parr 30 domain (amino acids 340?533) having guanine nucleotide, otherFeat=[]-->, belongsTo=parr 31 exchange factor (GEF) activity towards the GTPase Rab1 (Machner, otherFeat=[]-->, belongsTo=parr 32 & Isberg, 2007; Schoebel et al, 2009; Suh et al, 2010), a carboxy-, otherFeat=[]-->, belongsTo=parr 33 terminal lipid-binding domain and an amino-terminal domain of, otherFeat=[]-->, belongsTo=parr 34 unknown function. Immediately after transfer from the Legionella, otherFeat=[]-->, belongsTo=parr 35 bacterium into the host cytosol, DrrA localizes to the cytosolic, otherFeat=[]-->, belongsTo=parr 36 surface of the LCV through its C-terminal domain by binding, otherFeat=[]-->, belongsTo=parr 37 specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P;, otherFeat=[]-->, belongsTo=parr 38 Brombacher et al, 2009) . Once bound to the LCV, the GEF, otherFeat=[]-->, belongsTo=parr 39 domain of DrrA mediates redirection of ER-derived vesicles, otherFeat=[]-->, belongsTo=parr 40 through the recruitment and activation of Rab1 (Derre & Isberg,, otherFeat=[]-->, belongsTo=parr 41 2004; Kagan et al, 2004; Schoebel et al, 2009). As Rab proteins,, otherFeat=[]-->, belongsTo=parr 42 such as Rab1, can exert the function of regulating vesicular, otherFeat=[]-->, belongsTo=parr 43 transport only when they are attached to their respective target, otherFeat=[]-->, belongsTo=parr 44 membrane, the correct membrane localization of the Rab-, otherFeat=[]-->, belongsTo=parr 45 activating GEFs is crucial. Thus, Legionella ensures the correct, otherFeat=[]-->, belongsTo=parr 46 targeting and activation of Rab1 during infection to the LCV by, otherFeat=[]-->, belongsTo=parr 47 attaching DrrA through lipid binding to the LCV., otherFeat=[]-->, belongsTo=parr 48 The PtdIns(4)P-binding domain of SidM/DrrA (P4M) has been, otherFeat=[]-->, belongsTo=parr 49 identified recently (Brombacher et al, 2009). To understand the, otherFeat=[]-->, belongsTo=parr 50 structural basis for PtdIns(4)P recognition, we have determined, otherFeat=[]-->, belongsTo=parr 51 the crystal structure of P4M. A subsequent quantification revealed, otherFeat=[]-->, belongsTo=parr 52 an unusually high binding affinity for the P4M?PtdIns(4)P interaction,, otherFeat=[]-->, belongsTo=parr 53 which has not been observed for other phosphatidylinositol, otherFeat=[]-->, belongsTo=parr 54 phosphate (PtdInsP)-binding proteins., otherFeat=[]-->, belongsTo=parr 55 RESULTS AND DISCUSSION, otherFeat=['U']-->, belongsTo=title 56 Structure of DrrA340?647, otherFeat=[]-->, belongsTo=title 57 The minimal fragment of P4M is small, consisting of about 100, otherFeat=[]-->, belongsTo=parr 58 amino acids, and has no sequence homology to any known lipid-, otherFeat=[]-->, belongsTo=parr 59 binding domain. As constructs containing only P4M (amino acids, otherFeat=[]-->, belongsTo=parr 60 544?647) seemed to reduce PtdIns(4)P binding to some degree, otherFeat=[]-->, belongsTo=parr 61 (Brombacher et al, 2009), we determined the crystal structure of a, otherFeat=[]-->, belongsTo=parr 62 fragment of DrrA comprising the GEF domain and P4M (amino, otherFeat=[]-->, belongsTo=parr 63 acids 340?647) at 2.5 A? resolution (Fig 1A; for data collection,, otherFeat=[]-->, belongsTo=parr 64 phasing and refinement statistics, see Table 1). As reported, otherFeat=[]-->, belongsTo=parr 65 previously for the GEF domain (Schoebel et al, 2009; Suh et al,, otherFeat=[]-->, belongsTo=parr 66 2010), P4M has a new protein fold with no homologous structures, otherFeat=[]-->, belongsTo=parr 67 found in the Protein Data Bank when running a search using the, otherFeat=[]-->, belongsTo=parr 68 Dali server (Holm et al, 2008). P4M consists of approximately, otherFeat=[]-->, belongsTo=parr 69 50% of a-helices (six a-helices and one 310-helix) and 50% of, otherFeat=[]-->, belongsTo=parr 70 ordered loops. The three central helices aPI2, aPI3 and aPI6 are, otherFeat=[]-->, belongsTo=parr 71 arranged perpendicularly to a-helices aG6?aG8 of the GEF, otherFeat=[]-->, belongsTo=parr 72 domain. At the tip of these helices, two sulphate ions from the, otherFeat=[]-->, belongsTo=parr 73 crystallization buffer are found in a positively charged pocket, otherFeat=[]-->, belongsTo=parr 74 (Fig 1B), surrounded mainly by the aPI1?aPI2 loop and the, otherFeat=[]-->, belongsTo=parr 75 aPI4?aPI5 loop (Fig 1C). The sulphate ions are spaced by 7.2 A?,, otherFeat=[]-->, belongsTo=parr 76 in accordance with the distance between the phosphate groups in, otherFeat=[]-->, belongsTo=parr 77 PtdIns(4)P (Fig 1C, right). This mimicking property of sulphate ions, otherFeat=[]-->, belongsTo=parr 78 Received 20 February 2010; revised 9 June 2010; accepted 9 June 2010;, otherFeat=[]-->, belongsTo=parrnote 79 published online 9 July 2010, otherFeat=[]-->, belongsTo=parrnote 80 +Corresponding author. Tel: ? 49 231 1332300; Fax: ? 49 231 1332399;, otherFeat=[]-->, belongsTo=parrnote 81 E-mail: roger.goody@mpi-dortmund.mpg.de, otherFeat=[]-->, belongsTo=parrnote 82 ++Corresponding author. Tel: ? 49 231 1332305; Fax: ? 49 231 1332399;, otherFeat=[]-->, belongsTo=parrnote 83 E-mail: aymelt.itzen@mpi-dortmund.mpg.de, otherFeat=[]-->, belongsTo=parrnote 84 Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology,, otherFeat=[]-->, belongsTo=parrnote 85 Otto-Hahn-Strasse 11, Dortmund, North Rhine-Westphalia 44227, Germany, otherFeat=[]-->, belongsTo=parrnote 86 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 87 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 88 scientificreport, otherFeat=[]-->, belongsTo=title 89 scientific report, otherFeat=[]-->, belongsTo=title 90 598, otherFeat=[]-->, belongsTo=nota_cab_pie 91 for binding of the phosphate head groups of PtdInsP has been, otherFeat=[]-->, belongsTo=parr 92 observed in another instance (p47phox-PX; Karathanassis et al,, otherFeat=[]-->, belongsTo=parr 93 2002). It is therefore probable that this positively charged pocket, otherFeat=[]-->, belongsTo=parr 94 constitutes the binding site for the PtdIns(4)P head group, otherFeat=[]-->, belongsTo=parr 95 (supplementary Fig S1 online). Furthermore, a negatively charged, otherFeat=[]-->, belongsTo=parr 96 surface patch is observed on the opposite side of the presumed, otherFeat=[]-->, belongsTo=parr 97 PtdIns(4)P-binding cavity, which is likely to be repelled by the, otherFeat=[]-->, belongsTo=parr 98 negatively charged cytosolic surface of intracellular membranes, otherFeat=[]-->, belongsTo=parr 99 (Fig 1B). This effect will presumably help to orient the protein, otherFeat=[]-->, belongsTo=parr 100 during the LCV-binding process such that the supposed PtdIns(4)P-, otherFeat=[]-->, belongsTo=parr 101 binding pocket faces towards intracellular PtdIns(4)P-containing, otherFeat=[]-->, belongsTo=parr 102 membranes (Weber et al, 2006) ., otherFeat=[]-->, belongsTo=parr 103 The interactions between the P4M and GEF domains are, otherFeat=[]-->, belongsTo=parr 104 mainly polar (supplementary Fig S2 online), which leads to a, otherFeat=[]-->, belongsTo=parr 105 defined relative orientation that is probably also stable in solution, otherFeat=[]-->, belongsTo=parr 106 or when DrrA is bound at the membrane, as is corroborated by the, otherFeat=[]-->, belongsTo=parr 107 G4, otherFeat=[]-->, belongsTo=?? 108 G2, otherFeat=[]-->, belongsTo=?? 109 G1, otherFeat=[]-->, belongsTo=?? 110 G3, otherFeat=[]-->, belongsTo=?? 111 G6, otherFeat=[]-->, belongsTo=?? 112 N, otherFeat=['U']-->, belongsTo=?? 113 C PI6, otherFeat=[]-->, belongsTo=?? 114 G5, otherFeat=[]-->, belongsTo=?? 115 G8, otherFeat=[]-->, belongsTo=?? 116 G7, otherFeat=[]-->, belongsTo=?? 117 Pl2, otherFeat=[]-->, belongsTo=?? 118 Pl4, otherFeat=[]-->, belongsTo=?? 119 Pl5, otherFeat=[]-->, belongsTo=?? 120 310, otherFeat=[]-->, belongsTo=?? 121 Pl1, otherFeat=[]-->, belongsTo=?? 122 SO2? #14, otherFeat=[]-->, belongsTo=?? 123 120?, otherFeat=[]-->, belongsTo=?? 124 Tyr 532, otherFeat=[]-->, belongsTo=?? 125 Asp 565, otherFeat=[]-->, belongsTo=?? 126 Lys 568, otherFeat=[]-->, belongsTo=?? 127 Ser 620, otherFeat=[]-->, belongsTo=?? 128 Ser 621, otherFeat=[]-->, belongsTo=?? 129 Gln 608, otherFeat=[]-->, belongsTo=?? 130 Thr 612, otherFeat=[]-->, belongsTo=?? 131 Arg 541, otherFeat=[]-->, belongsTo=?? 132 ?10 kBT, otherFeat=[]-->, belongsTo=?? 133 10 kBT, otherFeat=[]-->, belongsTo=?? 134 SO2? #2, otherFeat=[]-->, belongsTo=?? 135 4, otherFeat=[]-->, belongsTo=?? 136 Pl3, otherFeat=[]-->, belongsTo=?? 137 His 543, otherFeat=[]-->, belongsTo=?? 138 SO2? #24, otherFeat=[]-->, belongsTo=?? 139 SO2? #14, otherFeat=[]-->, belongsTo=?? 140 ?2O4, otherFeat=[]-->, belongsTo=?? 141 OH, otherFeat=['U']-->, belongsTo=?? 142 7.2 ?, otherFeat=['U']-->, belongsTo=?? 143 Ser 621, otherFeat=[]-->, belongsTo=?? 144 Ser 620, otherFeat=[]-->, belongsTo=?? 145 Gln 608, otherFeat=[]-->, belongsTo=?? 146 Lys 568, otherFeat=[]-->, belongsTo=?? 147 Tyr 532 Arg 541 His 543, otherFeat=[]-->, belongsTo=?? 148 Asp 565, otherFeat=[]-->, belongsTo=?? 149 Thr 612, otherFeat=[]-->, belongsTo=?? 150 OH, otherFeat=['U']-->, belongsTo=?? 151 OH OH, otherFeat=['U']-->, belongsTo=?? 152 O, otherFeat=['U']-->, belongsTo=?? 153 O, otherFeat=['U']-->, belongsTo=?? 154 O, otherFeat=['U']-->, belongsTo=?? 155 O, otherFeat=['U']-->, belongsTo=?? 156 R, otherFeat=['U']-->, belongsTo=?? 157 P, otherFeat=['U']-->, belongsTo=?? 158 P, otherFeat=['U']-->, belongsTo=?? 159 SO2?, otherFeat=[]-->, belongsTo=?? 160 4, otherFeat=[]-->, belongsTo=?? 161 SO2?, otherFeat=[]-->, belongsTo=?? 162 4, otherFeat=[]-->, belongsTo=?? 163 A, otherFeat=[]-->, belongsTo=?? 164 B, otherFeat=['U']-->, belongsTo=?? 165 C, otherFeat=[]-->, belongsTo=?? 166 Fig 1 | DrrA reveals a new fold for phosphatidylinositol-4-phosphate binding. (A) Representation of the crystal structure of DrrA340?647. The GEF, otherFeat=[]-->, belongsTo=fig_caption 167 domain is shown in light blue, P4M in the spectrum from blue to red. Two sulphate ions (SO42? #1 and SO42? #2) found in the crystal structure, otherFeat=[]-->, belongsTo=fig_caption 168 indicate the presumed PtdIns(4)P-binding pocket and are drawn in stick representation. (B) Surface representations of P4M coloured by its, otherFeat=[]-->, belongsTo=fig_caption 169 electrostatic potential with two sulphate ions (stick representation) occupying the PtdIns(4)P-binding pocket. (C) Left: polar contacts between sulphate, otherFeat=[]-->, belongsTo=fig_caption 170 ions (sticks), the sulphate surrounding water molecules (red spheres) and selected residues of P4M. Right: schematic depiction of the polar contacts, otherFeat=[]-->, belongsTo=fig_caption 171 between DrrA and sulphate ions. The PtdIns(4)P head group is depicted schematically below to illustrate the spacing between 1- and 4-phosphates, otherFeat=[]-->, belongsTo=fig_caption 172 (sulphates are numbered according to Fig 1A). GEF, guanine nucleotide exchange factor; P4M, PtdIns(4)P-binding domain of SidM/DrrA;, otherFeat=[]-->, belongsTo=fig_caption 173 PtdIns(4)P, phosphatidylinositol 4-phosphate., otherFeat=[]-->, belongsTo=fig_caption 174 Molecular basis of PtdIns(4)P binding by DrrA, otherFeat=[]-->, belongsTo=nota_cab_pie 175 S. Schoebel et al, otherFeat=[]-->, belongsTo=nota_cab_pie 176 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 177 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 178 scientific report, otherFeat=[]-->, belongsTo=nota_cab_pie 179 59 9, otherFeat=[]-->, belongsTo=nota_cab_pie 180 crystal structure of a similar DrrA fragment that was published, otherFeat=[]-->, belongsTo=parr 181 while this paper was under review (Zhu et al, 2010). This structure, otherFeat=[]-->, belongsTo=parr 182 is nearly identical to the one described here (supplementary, otherFeat=[]-->, belongsTo=parr 183 Fig S3A online), despite having been obtained at markedly, otherFeat=[]-->, belongsTo=parr 184 different pH and from crystals that diffract to only 3.5 A?, whereas, otherFeat=[]-->, belongsTo=parr 185 our model was refined at 2.5 A?. The higher resolution allowed, otherFeat=[]-->, belongsTo=parr 186 us to identify the position of a second sulphate ion, which leads, otherFeat=[]-->, belongsTo=parr 187 to the modelling of the presumed position and orientation, otherFeat=[]-->, belongsTo=parr 188 of PtdIns(4)P, thereby providing a reasonable model for the, otherFeat=[]-->, belongsTo=parr 189 mechanism of PtdIns(4)P recognition (supplementary Fig S1 online), otherFeat=[]-->, belongsTo=parr 190 and membrane binding (see below). There is no structural similarity, otherFeat=[]-->, belongsTo=parr 191 between P4M and other PtdInsP-binding domains. However, the, otherFeat=[]-->, belongsTo=parr 192 Epsin N-terminal homology domain of Epsin is also an a-helical, otherFeat=[]-->, belongsTo=parr 193 protein, and harbours an amphiphatic a-helix (a0) close to the, otherFeat=[]-->, belongsTo=parr 194 PtdInsP-binding pocket that is implicated in membrane binding, otherFeat=[]-->, belongsTo=parr 195 (Ford et al, 2002). This is also seen in our structure (supplementary, otherFeat=[]-->, belongsTo=parr 196 Fig S3B online), supporting the idea that a-helix aPI5 could be, otherFeat=[]-->, belongsTo=parr 197 involved in membrane binding., otherFeat=[]-->, belongsTo=parr 198 Interaction of DrrA340?647 with PtdIns(4)P, otherFeat=[]-->, belongsTo=title 199 To determine the affinity between DrrA and PtdIns(4)P, we, otherFeat=[]-->, belongsTo=parr 200 performed isothermal titration calorimetry measurements with a, otherFeat=[]-->, belongsTo=parr 201 water-soluble analogue of PtdIns(4)P, namely, di-C4-PtdIns(4)P., otherFeat=[]-->, belongsTo=parr 202 The experiment revealed an unexpectedly high affinity of DrrA, otherFeat=[]-->, belongsTo=parr 203 towards di-C4-PtdIns(4)P, with a dissociation equilibrium constant, otherFeat=[]-->, belongsTo=parr 204 (KD) of approximately 30 nM (Fig 2A). In further experiments,, otherFeat=[]-->, belongsTo=parr 205 it was possible to determine the association and dissociation, otherFeat=[]-->, belongsTo=parr 206 rate constants (kon and koff, respectively) of DrrA340?647 by, otherFeat=[]-->, belongsTo=note 207 using a fluorescent analogue of di-C4-PtdIns(4)P labelled with, otherFeat=[]-->, belongsTo=parr 208 boron-dipyrromethene (BODIPY; BODIPY?PtdIns(4)P), exploiting, otherFeat=[]-->, belongsTo=parr 209 the change in fluorescence polarization on binding. Fitting the, otherFeat=[]-->, belongsTo=parr 210 association traces to single exponentials (Fig 2B) and plotting, otherFeat=[]-->, belongsTo=parr 211 the observed pseudo-first order rate constants against the DrrA, otherFeat=[]-->, belongsTo=parr 212 concentration resulted in kon ? 3.2 ? 106 M?1 s?1 (Fig 2C). In a, otherFeat=[]-->, belongsTo=parr 213 separate experiment, the displacement of BODIPY?PtdIns(4)P, otherFeat=[]-->, belongsTo=parr 214 from DrrA with non-fluorescent di-C4-PtdIns(4)P resulted in a, otherFeat=[]-->, belongsTo=parr 215 slow dissociation rate constant (koff) of 0.016 s?1 (Fig 2C, inset)., otherFeat=[]-->, belongsTo=parr 216 Together, the KD value for the DrrA?(BODIPY?PtdIns(4)P) interaction, otherFeat=[]-->, belongsTo=parr 217 is calculated to be 5 nM., otherFeat=[]-->, belongsTo=parr 218 As it is possible that the high binding affinity between DrrA, otherFeat=[]-->, belongsTo=parr 219 and BODIPY?PtdIns(4)P is in part due to the presence of the, otherFeat=[]-->, belongsTo=parr 220 fluorescence group, we performed competition experiments with, otherFeat=[]-->, belongsTo=parr 221 unlabelled di-C4-PtdIns(4)P (Fig 2D). The displacement of 200 nM, otherFeat=[]-->, belongsTo=parr 222 BODIPY?PtdIns(4)P from a mixture with 200 nM DrrA340?647, otherFeat=[]-->, belongsTo=parr 223 was monitored by the addition of 400 nM di-C4-PtdIns(4)P, otherFeat=[]-->, belongsTo=parr 224 and was fitted to a simple competition model. From the end point, otherFeat=[]-->, belongsTo=parr 225 of the reaction, the dissociation constant for di-C4-PtdIns(4)P was, otherFeat=[]-->, belongsTo=parr 226 determined to be KD ? 18.2 nM. Kinetic constants were calculated, otherFeat=[]-->, belongsTo=parr 227 from the time dependence of the change in fluorescence, otherFeat=[]-->, belongsTo=parr 228 polarization (kon ? 4.1 ? 106 M?1 s?1, koff ? 0.079 s?1). Thus, the, otherFeat=[]-->, belongsTo=parr 229 absence of the fluorescence reporter group resulted in a fourfold, otherFeat=[]-->, belongsTo=parr 230 weaker di-C4-PtdIns(4)P binding compared with BODIPY?, otherFeat=[]-->, belongsTo=parr 231 PtdIns(4)P. Nevertheless, to the best of our knowledge, the affinity, otherFeat=[]-->, belongsTo=parr 232 of P4M-PtdIns(4)P is at least an order of magnitude higher than, otherFeat=[]-->, belongsTo=parr 233 that of most PtdInsP?protein interactions reported so far. The, otherFeat=[]-->, belongsTo=parr 234 strongest interaction with a PtdInsP known to date is between, otherFeat=[]-->, belongsTo=parr 235 the PH domains of oxysterol-binding protein and PtdIns(4)P and, otherFeat=[]-->, belongsTo=parr 236 has a KD value of 40?100 nM (Stahelin et al, 2007) ., otherFeat=[]-->, belongsTo=parr 237 Owing to the close proximity of the P4M and GEF domains of, otherFeat=[]-->, belongsTo=parr 238 DrrA in the crystal structure, we investigated whether Rab1b can, otherFeat=[]-->, belongsTo=parr 239 modulate the affinity of DrrA towards PtdIns(4)P. For this purpose,, otherFeat=[]-->, belongsTo=parr 240 we measured the time-dependent displacement of BODIPY?, otherFeat=[]-->, belongsTo=parr 241 PtdIns(4)P from DrrA340?647 with unlabelled PtdIns(4)P in the, otherFeat=[]-->, belongsTo=parr 242 presence of Rab1b:GDP and Rab1b:GTP (supplementary Fig S4, otherFeat=[]-->, belongsTo=parr 243 online). The rate of dissociation of BODIPY?PtdIns(4)P from the, otherFeat=[]-->, belongsTo=parr 244 complex was unchanged, regardless of the presence of Rab1b:, otherFeat=[]-->, belongsTo=parr 245 GDP, Rab1b:GTP or the nucleotide-free Rab1b:DrrA340?647, otherFeat=[]-->, belongsTo=parr 246 complex, indicating that Rab1b has no modulatory role on, otherFeat=[]-->, belongsTo=parr 247 PtdIns(4)P affinity for P4M., otherFeat=[]-->, belongsTo=parr 248 Using the fluorescent binding assay, we also investigated the, otherFeat=[]-->, belongsTo=parr 249 relevance of the positively charged pocket of P4M of DrrA for, otherFeat=[]-->, belongsTo=parr 250 PtdIns(4)P binding. As the sulphate ions could potentially mimic, otherFeat=[]-->, belongsTo=parr 251 the position of the 4-phosphate head group of PtdIns(4)P, we, otherFeat=[]-->, belongsTo=parr 252 mutated the sulphate-interacting residue Lys 568 to alanine, otherFeat=[]-->, belongsTo=parr 253 (Fig 1C). Melting point analysis by circular dichroism showed no, otherFeat=[]-->, belongsTo=parr 254 effect on protein stability (supplementary Fig S5 online), but the, otherFeat=[]-->, belongsTo=parr 255 mutation completely abolished the DrrA?(BODIPY?PtdIns(4)P), otherFeat=[]-->, belongsTo=parr 256 interaction (Fig 2E). This result confirms that the positively charged, otherFeat=[]-->, belongsTo=parr 257 Table 1 | Data collection, phasing and refinement statistics, otherFeat=[]-->, belongsTo=parr 258 DrrA340?647, otherFeat=[]-->, belongsTo=parrnote 259 DrrA340?647 SeMet*, otherFeat=[]-->, belongsTo=note 260 Data collectionz, otherFeat=[]-->, belongsTo=parrnote 261 Space group, otherFeat=[]-->, belongsTo=parrnote 262 P212121, otherFeat=[]-->, belongsTo=parrnote 263 P212121, otherFeat=[]-->, belongsTo=parrnote 264 Cell dimensions, otherFeat=[]-->, belongsTo=parrnote 265 a, b, c (A? ), otherFeat=[]-->, belongsTo=parrnote 266 74.9, 75.4, 131.0, otherFeat=[]-->, belongsTo=parrnote 267 74.1, 75.3, 131.0, otherFeat=[]-->, belongsTo=parrnote 268 a, b, g ( 1), otherFeat=[]-->, belongsTo=parrnote 269 90, 90, 90, otherFeat=[]-->, belongsTo=parrnote 270 90, 90, 90, otherFeat=[]-->, belongsTo=parrnote 271 Resolution (A? )y, otherFeat=[]-->, belongsTo=parrnote 272 20?2.5 (2.6?2.5), otherFeat=[]-->, belongsTo=parrnote 273 20?2.7 (2.8?2.7), otherFeat=[]-->, belongsTo=parrnote 274 Rmean, otherFeat=[]-->, belongsTo=parrnote 275 11.1 (41.2), otherFeat=[]-->, belongsTo=parrnote 276 15.6 (46.2), otherFeat=[]-->, belongsTo=parrnote 277 I/sI, otherFeat=[]-->, belongsTo=parrnote 278 15.8 (4.2), otherFeat=[]-->, belongsTo=parrnote 279 10.6 (4.0), otherFeat=[]-->, belongsTo=parrnote 280 Completeness (%), otherFeat=[]-->, belongsTo=parrnote 281 100 (100), otherFeat=[]-->, belongsTo=parrnote 282 100 (100), otherFeat=[]-->, belongsTo=parrnote 283 Redundancy, otherFeat=[]-->, belongsTo=parrnote 284 8.1 (8.2), otherFeat=[]-->, belongsTo=parrnote 285 7.3 (7.1), otherFeat=[]-->, belongsTo=parrnote 286 Refinement, otherFeat=[]-->, belongsTo=parrnote 287 Resolution (A? ), otherFeat=[]-->, belongsTo=parrnote 288 2.5, otherFeat=[]-->, belongsTo=parrnote 289 No. of reflections, otherFeat=[]-->, belongsTo=parrnote 290 26,298, otherFeat=[]-->, belongsTo=parrnote 291 Rwork/Rfree, otherFeat=[]-->, belongsTo=parrnote 292 18.9/26.2, otherFeat=[]-->, belongsTo=parrnote 293 No. of atoms, otherFeat=[]-->, belongsTo=parrnote 294 Protein, otherFeat=[]-->, belongsTo=parrnote 295 4,752, otherFeat=[]-->, belongsTo=parrnote 296 Ligand/ion, otherFeat=[]-->, belongsTo=parrnote 297 40, otherFeat=[]-->, belongsTo=parrnote 298 Water, otherFeat=[]-->, belongsTo=parrnote 299 174, otherFeat=[]-->, belongsTo=parrnote 300 B-factors, otherFeat=[]-->, belongsTo=parrnote 301 Protein, otherFeat=[]-->, belongsTo=parrnote 302 35, otherFeat=[]-->, belongsTo=parrnote 303 Ligand/ion, otherFeat=[]-->, belongsTo=parrnote 304 42, otherFeat=[]-->, belongsTo=parrnote 305 Water, otherFeat=[]-->, belongsTo=parrnote 306 40, otherFeat=[]-->, belongsTo=parrnote 307 r.m.s.d.J, otherFeat=[]-->, belongsTo=parrnote 308 Bond lengths (A? ), otherFeat=[]-->, belongsTo=parrnote 309 0.019, otherFeat=[]-->, belongsTo=parrnote 310 Bond angles ( 1), otherFeat=[]-->, belongsTo=parrnote 311 1.658, otherFeat=[]-->, belongsTo=parrnote 312 *Data collection statistics for single-wavelength anomalous dispersion data, otherFeat=[]-->, belongsTo=parrnote 313 refer to unmerged Friedel pairs; zdata sets were collected from a single crystal;, otherFeat=[]-->, belongsTo=parrnote 314 yvalues in parentheses refer to the highest resolution shell; Jr.m.s.d. values., otherFeat=[]-->, belongsTo=parrnote 315 Molecular basis of PtdIns(4)P binding by DrrA, otherFeat=[]-->, belongsTo=nota_cab_pie 316 S. Schoebel et al, otherFeat=[]-->, belongsTo=nota_cab_pie 317 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 318 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 319 scientific report, otherFeat=[]-->, belongsTo=nota_cab_pie 320 600, otherFeat=[]-->, belongsTo=nota_cab_pie 321 pocket of P4M containing sulphates is indeed the binding cavity, otherFeat=[]-->, belongsTo=parr 322 for PtdIns(4)P., otherFeat=[]-->, belongsTo=parr 323 Biological relevance of the high affinity of PtdIns(4)P, otherFeat=[]-->, belongsTo=title 324 To recruit Rab1, DrrA must associate with LCV after it has been, otherFeat=[]-->, belongsTo=parr 325 released into the cytosol of the host cell. PtdIns(4)P is found, otherFeat=[]-->, belongsTo=parr 326 mainly in the Golgi and to a lesser extent in the plasma membrane, otherFeat=[]-->, belongsTo=parr 327 (di Paolo & de Camilli, 2006), the latter being the primary origin, otherFeat=[]-->, belongsTo=parr 328 of LCV. Owing to the presumably low abundance of PtdIns(4)P in, otherFeat=[]-->, belongsTo=parr 329 the LCV immediately after Legionella phagocytosis and because of, otherFeat=[]-->, belongsTo=parr 330 possible competition with other PtdIns(4)P-binding proteins, DrrA, otherFeat=[]-->, belongsTo=parr 331 seems to have evolved to bind to PtdIns(4)P with exceptionally, otherFeat=[]-->, belongsTo=parr 332 high affinity. DrrA is released from the LCV in the course of the, otherFeat=[]-->, belongsTo=parr 333 Legionella infectious cycle, so that membrane binding is only, otherFeat=[]-->, belongsTo=parr 334 transient (Ingmundson et al, 2007) . As binding towards PtdIns(4)P, otherFeat=[]-->, belongsTo=parr 335 is exceptionally strong, release from the membrane most probably, otherFeat=[]-->, belongsTo=parr 336 occurs as a result of a reduction of the amount of PtdIns(4)P in the, otherFeat=[]-->, belongsTo=parr 337 course of the maturation of LCVs. Despite the high affinity, the, otherFeat=[]-->, belongsTo=parr 338 DrrA:PtdIns(4)P complex is moderately dynamic (koff ? 0.079 s?1;, otherFeat=[]-->, belongsTo=note 339 that is, half-life ? ln2/koff ? 8.7 s) and would therefore allow for, otherFeat=[]-->, belongsTo=parr 340 PtdIns(4)P modifications on a physiologically relevant time scale., otherFeat=[]-->, belongsTo=parr 341 The high specificity of DrrA for PtdIns(4)P (Brombacher et al,, otherFeat=[]-->, belongsTo=parr 342 2009), together with its exceptionally high binding affinity, makes, otherFeat=[]-->, belongsTo=parr 343 P4M a potentially valuable molecular tool for the specific and, otherFeat=[]-->, belongsTo=parr 344 sensitive labelling of intracellular membranes for the presence of, otherFeat=[]-->, belongsTo=parr 345 PtdIns(4)P. As proof of principle, it was shown that the PH, otherFeat=[]-->, belongsTo=parr 346 domains of oxysterol-binding protein and four-phosphate adaptor, otherFeat=[]-->, belongsTo=parr 347 protein 1 can be used to monitor the cellular activity and spatial, otherFeat=[]-->, belongsTo=parr 348 distribution of the PtdIns(4)P-generating enzyme PtdIns(4) kinase, otherFeat=[]-->, belongsTo=parr 349 (Balla et al, 2005). However, the tighter interaction of DrrA with, otherFeat=[]-->, belongsTo=parr 350 PtdIns(4)P makes the protein potentially even better suited to, otherFeat=[]-->, belongsTo=parr 351 this purpose., otherFeat=[]-->, belongsTo=parr 352 0, otherFeat=[]-->, belongsTo=parrnote 353 020, otherFeat=[]-->, belongsTo=parrnote 354 0, otherFeat=[]-->, belongsTo=parrnote 355 0, otherFeat=[]-->, belongsTo=parrnote 356 50 100 150, otherFeat=[]-->, belongsTo=parrnote 357 Time (ms), otherFeat=[]-->, belongsTo=parrnote 358 Time (s), otherFeat=[]-->, belongsTo=parrnote 359 Time (s), otherFeat=[]-->, belongsTo=parrnote 360 DrrA340?647 K568A, otherFeat=[]-->, belongsTo=parrnote 361 DrrA 340?647, otherFeat=[]-->, belongsTo=parrnote 362 DrrA340?647 (M), otherFeat=[]-->, belongsTo=parrnote 363 DrrAfl, otherFeat=[]-->, belongsTo=parrnote 364 200, otherFeat=[]-->, belongsTo=parrnote 365 0, otherFeat=[]-->, belongsTo=parrnote 366 0.5, otherFeat=[]-->, belongsTo=parrnote 367 1.0, otherFeat=[]-->, belongsTo=parrnote 368 0.5, otherFeat=[]-->, belongsTo=parrnote 369 N = 1.3, otherFeat=['U']-->, belongsTo=parrnote 370 KD = 30 nM, otherFeat=[]-->, belongsTo=parrnote 371 H = 29,240 cal mol?1, otherFeat=[]-->, belongsTo=parrnote 372 S = ?64 cal mol?1 K?1, otherFeat=[]-->, belongsTo=parrnote 373 1.0 1.5, otherFeat=[]-->, belongsTo=parrnote 374 Molar ratio, otherFeat=[]-->, belongsTo=parrnote 375 2.0 2.5, otherFeat=[]-->, belongsTo=parrnote 376 0, otherFeat=[]-->, belongsTo=parrnote 377 100 200 300 400 500, otherFeat=[]-->, belongsTo=parrnote 378 6.5 M, otherFeat=[]-->, belongsTo=parrnote 379 3.25 M, otherFeat=[]-->, belongsTo=parrnote 380 1.63 M, otherFeat=[]-->, belongsTo=parrnote 381 0.81 M, otherFeat=[]-->, belongsTo=parrnote 382 0.40 M, otherFeat=[]-->, belongsTo=parrnote 383 13 M, otherFeat=[]-->, belongsTo=parrnote 384 40, otherFeat=[]-->, belongsTo=parrnote 385 60, otherFeat=[]-->, belongsTo=parrnote 386 Time (min), otherFeat=[]-->, belongsTo=parrnote 387 cal, otherFeat=[]-->, belongsTo=parrnote 388 s, otherFeat=[]-->, belongsTo=parrnote 389 ?1, otherFeat=[]-->, belongsTo=parrnote 390 kcal, otherFeat=[]-->, belongsTo=parrnote 391 per, otherFeat=[]-->, belongsTo=parrnote 392 mole, otherFeat=[]-->, belongsTo=parrnote 393 of, otherFeat=[]-->, belongsTo=parrnote 394 injectant, otherFeat=[]-->, belongsTo=parrnote 395 FP, otherFeat=['U']-->, belongsTo=parrnote 396 (mP), otherFeat=[]-->, belongsTo=parrnote 397 FP, otherFeat=['U']-->, belongsTo=parrnote 398 (mP), otherFeat=[]-->, belongsTo=parrnote 399 FP, otherFeat=['U']-->, belongsTo=parrnote 400 (mP), otherFeat=[]-->, belongsTo=parrnote 401 ?1, otherFeat=[]-->, belongsTo=parrnote 402 ?2, otherFeat=[]-->, belongsTo=parrnote 403 ?10, otherFeat=[]-->, belongsTo=parrnote 404 ?20, otherFeat=[]-->, belongsTo=parrnote 405 ?30, otherFeat=[]-->, belongsTo=parrnote 406 200, otherFeat=[]-->, belongsTo=parrnote 407 150, otherFeat=[]-->, belongsTo=parrnote 408 100, otherFeat=[]-->, belongsTo=parrnote 409 50, otherFeat=[]-->, belongsTo=parrnote 410 0, otherFeat=[]-->, belongsTo=parrnote 411 200, otherFeat=[]-->, belongsTo=parrnote 412 100, otherFeat=[]-->, belongsTo=parrnote 413 120, otherFeat=[]-->, belongsTo=parrnote 414 140, otherFeat=[]-->, belongsTo=parrnote 415 160, otherFeat=[]-->, belongsTo=parrnote 416 150, otherFeat=[]-->, belongsTo=parrnote 417 100, otherFeat=[]-->, belongsTo=parrnote 418 50, otherFeat=[]-->, belongsTo=parrnote 419 0, otherFeat=[]-->, belongsTo=parrnote 420 0, otherFeat=[]-->, belongsTo=parrnote 421 ?3, otherFeat=[]-->, belongsTo=parrnote 422 A, otherFeat=[]-->, belongsTo=parrnote 423 D, otherFeat=['U']-->, belongsTo=parrnote 424 E, otherFeat=['U']-->, belongsTo=parrnote 425 B, otherFeat=['U']-->, belongsTo=parrnote 426 Time (s), otherFeat=[]-->, belongsTo=parrnote 427 k obs, otherFeat=[]-->, belongsTo=parrnote 428 (s, otherFeat=[]-->, belongsTo=parrnote 429 ?1 ), otherFeat=[]-->, belongsTo=parrnote 430 0, otherFeat=[]-->, belongsTo=parrnote 431 5 10 15, otherFeat=[]-->, belongsTo=parrnote 432 500, otherFeat=[]-->, belongsTo=parrnote 433 250, otherFeat=[]-->, belongsTo=parrnote 434 0, otherFeat=[]-->, belongsTo=parrnote 435 FP, otherFeat=['U']-->, belongsTo=parrnote 436 (mP), otherFeat=[]-->, belongsTo=parrnote 437 40, otherFeat=[]-->, belongsTo=parrnote 438 30, otherFeat=[]-->, belongsTo=parrnote 439 20, otherFeat=[]-->, belongsTo=parrnote 440 10, otherFeat=[]-->, belongsTo=parrnote 441 0, otherFeat=[]-->, belongsTo=parrnote 442 150, otherFeat=[]-->, belongsTo=parrnote 443 100, otherFeat=[]-->, belongsTo=parrnote 444 0, otherFeat=[]-->, belongsTo=parrnote 445 C, otherFeat=[]-->, belongsTo=parrnote 446 Fig 2 | DrrA binds phosphatidylinositol 4-phosphate selectively with unexpectedly high affinity. (A) Isothermal titration calorimetry experiment of, otherFeat=[]-->, belongsTo=parrnote 447 50 mM di-C4-PtdIns(4)P titrated into 5 mM DrrA340?647.(B) Individual stopped-flow time traces of the association between 166 nM BODIPY?PtdIns(4)P, otherFeat=[]-->, belongsTo=parrnote 448 and indicated concentrations of DrrA340?647 monitored by the change in fluorescence polarization. (C) Linear fit of the observed DrrA340?647?BODIPY?, otherFeat=[]-->, belongsTo=parrnote 449 PtdIns(4)P association rates against DrrA340?647 concentration. Inset: BODIPY?PtdIns(4)P displacement from a DrrA340?647:BODIPY?PtdIns(4)P, otherFeat=[]-->, belongsTo=parrnote 450 complex (0.4 mM) by 5 mM di-C4-PtdIns(4)P. (D) Stopped-flow competition binding experiment (red line) using 200 nM DrrA340?647, 200 nM, otherFeat=[]-->, belongsTo=parrnote 451 BODIPY?PtdIns(4)P and 400 nM di-C4-PtdIns(4)P. The data were fitted to a simple competition model for the determination of KD, kon and koff, otherFeat=[]-->, belongsTo=parrnote 452 of di-C4-PtdIns(4)P to DrrA340?647.(E) Association of 166 nM BODIPY?PtdIns(4)P with 6.5 mM DrrAfl, DrrA340?647 or DrrA340?647 K568A., otherFeat=[]-->, belongsTo=parrnote 453 BODIPY, boron-dipyrromethene; FP, fluorescence polarization; PtdIns(4)P, phosphatidylinositol 4-phosphate., otherFeat=[]-->, belongsTo=parrnote 454 Molecular basis of PtdIns(4)P binding by DrrA, otherFeat=[]-->, belongsTo=nota_cab_pie 455 S. Schoebel et al, otherFeat=[]-->, belongsTo=nota_cab_pie 456 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 457 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 458 scientific report, otherFeat=[]-->, belongsTo=nota_cab_pie 459 60 1, otherFeat=[]-->, belongsTo=nota_cab_pie 460 The structure of DrrA340?647, comprising the GEF domain and, otherFeat=[]-->, belongsTo=parr 461 the P4M, allows us to propose a structural model for DrrA, otherFeat=[]-->, belongsTo=parr 462 membrane binding and Rab1 membrane anchoring (Fig 3)., otherFeat=[]-->, belongsTo=parr 463 DrrA presumably binds to PtdIns(4)P-containing membranes in a, otherFeat=[]-->, belongsTo=parr 464 manner that orients the GEF domain away from the membrane, otherFeat=[]-->, belongsTo=parr 465 surface. In this model, P4M can potentially interact with a, otherFeat=[]-->, belongsTo=parr 466 negatively charged membrane through an arginine residue of the, otherFeat=[]-->, belongsTo=parr 467 aPI1?aPI2 loop (Arg 544) and through hydrophobic interactions of, otherFeat=[]-->, belongsTo=parr 468 two leucines of the a-helix aPI5 (Leu 610, Leu 614) with the lipid, otherFeat=[]-->, belongsTo=parr 469 bilayer. The GEF domain extends into the cytosol and is able to, otherFeat=[]-->, belongsTo=parr 470 recruit Rab1 from a pool of cytosolic Rab1:GDI (GDP dissociation, otherFeat=[]-->, belongsTo=parr 471 inhibitor) complexes. Binding of Rab1 and the concomitant, otherFeat=[]-->, belongsTo=parr 472 nucleotide exchange from GDP to GTP are necessary for effective, otherFeat=[]-->, belongsTo=parr 473 GDP dissociation inhibitor displacement (Schoebel et al, 2009). In, otherFeat=[]-->, belongsTo=parr 474 our model, the binding of Rab1 to membrane-bound DrrA would, otherFeat=[]-->, belongsTo=parr 475 orient the GTPase with the nucleotide-binding pocket facing the, otherFeat=[]-->, belongsTo=parr 476 cytosol, thus allowing direct access for GTP to the Rab1:DrrA, otherFeat=[]-->, belongsTo=parr 477 complex. In addition, the C-terminus of Rab1 will be positioned in, otherFeat=[]-->, belongsTo=parr 478 close proximity to the membrane and could thus facilitate the, otherFeat=[]-->, belongsTo=parr 479 incorporation of the C-terminal geranylgeranyl moieties of Rab1, otherFeat=[]-->, belongsTo=parr 480 into the lipid bilayer. Thus, the P4M of DrrA achieves three goals, otherFeat=[]-->, belongsTo=parr 481 simultaneously: first, it binds specifically and tightly to PtdIns(4)P, otherFeat=[]-->, belongsTo=parr 482 and hence localizes DrrA to the LCV. Second, it orients the GEF, otherFeat=[]-->, belongsTo=parr 483 domain of DrrA in a manner that promotes undisturbed nucleotide, otherFeat=[]-->, belongsTo=parr 484 exchange on Rab1. Third, P4M brings the prenylated C-terminus, otherFeat=[]-->, belongsTo=parr 485 of Rab1 in close proximity to the membrane, thereby supporting, otherFeat=[]-->, belongsTo=parr 486 the insertion of geranylgeranyl moieties and leading to stable, otherFeat=[]-->, belongsTo=parr 487 attachment of Rab1 to the LCV for subsequent recruitment of, otherFeat=[]-->, belongsTo=parr 488 ER-derived vesicles., otherFeat=[]-->, belongsTo=parr 489 METHODS, otherFeat=['U']-->, belongsTo=title 490 Materials. Di-C4-PtdIns(4)P (di-C4-PtdIns(4)P, P-4004) and BODIPY, otherFeat=[]-->, belongsTo=parr 491 tetramethylrhodamine (TMR) PtdIns(4)P (BODIPY?PtdIns(4)P, C-04M6), otherFeat=[]-->, belongsTo=parr 492 were obtained from Echelon., otherFeat=[]-->, belongsTo=parr 493 Purification of Legionella DrrA340?647 and mutants. DrrA340?647, otherFeat=[]-->, belongsTo=parr 494 was subcloned into a modified pET19 vector (Novagen) that, otherFeat=[]-->, belongsTo=parr 495 contained an N-terminal hexa-histidine tag and a tobacco etch, otherFeat=[]-->, belongsTo=parr 496 virus (TEV) protease cleavage sequence. The site-specific mutant, otherFeat=[]-->, belongsTo=parr 497 DrrA340?647 K568A was created by using the QuikChange, otherFeat=[]-->, belongsTo=parr 498 Site-Directed Mutagenesis Kit (Stratagene). Proteins were, otherFeat=[]-->, belongsTo=parr 499 expressed in Escherichia coli (BL21-CodonPlus(DE3)-RIL) at 37 1C, otherFeat=[]-->, belongsTo=parr 500 after induction with 1 mM isopropyl-b-dithiogalactopyranoside., otherFeat=[]-->, belongsTo=parr 501 Selenomethionine-labelled DrrA340?647 was expressed using the, otherFeat=[]-->, belongsTo=parr 502 methionine biosynthesis inhibition method (van Duyne et al,, otherFeat=[]-->, belongsTo=parr 503 1993). After bacterial cell lysis, the supernatant was applied, otherFeat=[]-->, belongsTo=parr 504 to nickel?nitrilotriacetic acid (Ni?NTA) chromatography and, otherFeat=[]-->, belongsTo=parr 505 proteins were eluted with a linear Imidazole gradient (5?500 mM), otherFeat=[]-->, belongsTo=parr 506 in buffer A (20 mM HEPES (pH 8.0), 50 mM NaCl and 1 mM, otherFeat=[]-->, belongsTo=parr 507 DrrA-GEF, otherFeat=[]-->, belongsTo=parrnote 508 Nucleotide binding site, otherFeat=[]-->, belongsTo=parrnote 509 Rab1, otherFeat=[]-->, belongsTo=parrnote 510 N, otherFeat=['U']-->, belongsTo=parrnote 511 Prenyl moieties, otherFeat=[]-->, belongsTo=parrnote 512 ?, otherFeat=[]-->, belongsTo=parrnote 513 DrrA amino-terminal domain, otherFeat=[]-->, belongsTo=parrnote 514 C, otherFeat=[]-->, belongsTo=parrnote 515 DrrA-P4M, otherFeat=[]-->, belongsTo=parrnote 516 Ptdlns(4)P, otherFeat=[]-->, belongsTo=note 517 Membrane, otherFeat=[]-->, belongsTo=parrnote 518 Fig 3 | Structural model of DrrA membrane binding and simultaneous Rab1 interaction. The model demonstrates a possible orientation of P4M towards, otherFeat=[]-->, belongsTo=parrnote 519 a membrane and the implications for Rab1 binding to the DrrA?GEF domain. In this view, P4M orients the preceding GEF domain such that the, otherFeat=[]-->, belongsTo=parrnote 520 GDP/GTP-binding pocket of Rab1 points to the cytosol. Concomitantly, the carboxyl terminus of the GTPase is positioned in close proximity to, otherFeat=[]-->, belongsTo=parrnote 521 the membrane, thereby presumably facilitating the incorporation of the prenyl moieties into the LCV membrane. The position of Rab1 on DrrA340?647, otherFeat=[]-->, belongsTo=parrnote 522 was modelled by superimposing DrrA340?647 with the Rab13?174:DrrA340?533 complex structure (Schoebel et al, 2009). Green, DrrA-P4M; light blue,, otherFeat=[]-->, belongsTo=parrnote 523 DrrA-GEF domain; grey sphere, DrrA amino-terminal domain; orange, Rab1; ball and sticks, PtdIns(4)P model; dashed circle, GDP/GTP bindings site, otherFeat=[]-->, belongsTo=parrnote 524 of Rab1; red bars, schematic for Rab1 C-terminal prenyl moieties. GEF, guanine nucleotide exchange factor; LCV, Legionella-containing vacuole;, otherFeat=[]-->, belongsTo=parrnote 525 P4M, PtdIns(4)P-binding domain of SidM/DrrA; PtdIns(4)P, phosphatidylinositol 4-phosphate., otherFeat=[]-->, belongsTo=parrnote 526 Molecular basis of PtdIns(4)P binding by DrrA, otherFeat=[]-->, belongsTo=nota_cab_pie 527 S. Schoebel et al, otherFeat=[]-->, belongsTo=nota_cab_pie 528 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 529 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 530 scientific report, otherFeat=[]-->, belongsTo=nota_cab_pie 531 602, otherFeat=[]-->, belongsTo=nota_cab_pie 532 b-mercaptoethanol). Fractions containing DrrA340?647 or the, otherFeat=[]-->, belongsTo=parr 533 mutant protein were pooled and digested with His10-tagged, otherFeat=[]-->, belongsTo=parr 534 TEV protease while dialysing against buffer A to remove, otherFeat=[]-->, belongsTo=parr 535 imidazole. Uncleaved protein and TEV protease were removed, otherFeat=[]-->, belongsTo=parr 536 by passing over the Ni?NTA column again, and the concentrated, otherFeat=[]-->, belongsTo=parr 537 sample was subjected to size exclusion chromatography, otherFeat=[]-->, belongsTo=parr 538 (Superdex75 16/60, GE Healthcare) in buffer containing 20 mM, otherFeat=[]-->, belongsTo=parr 539 HEPES (pH 8.0), 50 mM NaCl and 2 mM dithiothreitol. The, otherFeat=[]-->, belongsTo=parr 540 protein was concentrated to 20 mg/ml and flash-frozen in liquid, otherFeat=[]-->, belongsTo=parr 541 nitrogen. Rab1b purification has been described previously, otherFeat=[]-->, belongsTo=parr 542 (Schoebel et al, 2009) ., otherFeat=[]-->, belongsTo=parr 543 Crystallization and structure determination of DrrA340?647. Native, otherFeat=[]-->, belongsTo=parr 544 and selenomethionine-labelled crystals of DrrA340?647 were, otherFeat=[]-->, belongsTo=parr 545 obtained by the hanging-drop vapour diffusion method by mixing, otherFeat=[]-->, belongsTo=parr 546 1 ml of protein (20 mg/ml) in gel filtration buffer with 1 mlof, otherFeat=[]-->, belongsTo=parr 547 a reservoir solution containing 12% (w/v) polyethylene glycol, otherFeat=[]-->, belongsTo=parr 548 4000, 20% (v/v) glycerol, 0.16 M ammonium sulphate and 0.1 M, otherFeat=[]-->, belongsTo=parr 549 sodium acetate at pH 4.8. Diffraction data were collected on, otherFeat=[]-->, belongsTo=parr 550 beamline X10SA of the Swiss Light Source and processed with, otherFeat=[]-->, belongsTo=parr 551 XDS (Kabsch, 1993 ). Selenium atoms were located and a model, otherFeat=[]-->, belongsTo=parr 552 was obtained with autoSHARP (Vonrhein et al, 2007) in MIR(AS), otherFeat=[]-->, belongsTo=parr 553 mode. Structure refinement was completed by using phenix.refine, otherFeat=[]-->, belongsTo=parr 554 (Adams et al, 2007) and manual rebuilding in Coot (Emsley &, otherFeat=[]-->, belongsTo=parr 555 Cowtan, 2004 ) with alternating rounds of refinement in REFMAC5, otherFeat=[]-->, belongsTo=parr 556 (Murshudov et al, 1997 ). The protein crystallizes in space group, otherFeat=[]-->, belongsTo=parr 557 P212121 with two independent copies of DrrA340?647 in the, otherFeat=[]-->, belongsTo=parr 558 asymmetric unit. No electron density is visible for C-terminal, otherFeat=[]-->, belongsTo=parr 559 residues 639?647. Molecular presentations were prepared with, otherFeat=[]-->, belongsTo=parr 560 PyMOL (Delano, 2002) ., otherFeat=[]-->, belongsTo=parr 561 Isothermal titration calorimetry of DrrA?PtdIns(4)P interaction., otherFeat=[]-->, belongsTo=parr 562 Di-C4-PtdIns(4)P (50 mM) in running buffer (20 mM HEPES (pH 8.0),, otherFeat=[]-->, belongsTo=parr 563 50 mM NaCl, 1 mM tris (2-carboxyethyl) phosphine) was titrated, otherFeat=[]-->, belongsTo=parr 564 automatically into a 5 mMsolution of DrrA340?647 dialysed in the, otherFeat=[]-->, belongsTo=parr 565 same buffer, using an iTC200 Micro Calorimeter (MicroCal)., otherFeat=[]-->, belongsTo=parr 566 Titrations were performed at 25 1C, with an injection volume of, otherFeat=[]-->, belongsTo=parr 567 6 ml at time intervals of 5 min to ensure that the titration peak, otherFeat=[]-->, belongsTo=parr 568 returned to baseline. Data were collected using iTC200 control, otherFeat=[]-->, belongsTo=parr 569 software and analysed with Origin (Version 7.0, MicroCal). Data, otherFeat=[]-->, belongsTo=parr 570 were corrected for heat of dilution of Di-C4-PtdIns(4)P into buffer,, otherFeat=[]-->, belongsTo=parr 571 which was determined in a separate titration., otherFeat=[]-->, belongsTo=parr 572 Kinetic analysis of PtdIns(4)P interaction with DrrA. All mea-, otherFeat=[]-->, belongsTo=parr 573 surements were carried out at 25 1C in buffer that contained, otherFeat=[]-->, belongsTo=parr 574 20 mM HEPES (pH 8.0), 50 mM NaCl and 2 mM dithiothreitol., otherFeat=[]-->, belongsTo=parr 575 Fast kinetic measurements were performed with a stopped-, otherFeat=[]-->, belongsTo=parr 576 flow apparatus (Applied Photophysics). For BODIPY TMR, otherFeat=[]-->, belongsTo=parr 577 PtdIns(4)P binding or release, time-dependent changes in fluores-, otherFeat=[]-->, belongsTo=parr 578 cence polarization were monitored with a 570nm cutoff filter, otherFeat=[]-->, belongsTo=parr 579 in the stopped-flow machine, excited at 546 nm. The competition, otherFeat=[]-->, belongsTo=parr 580 experiment to determine the affinity of non-fluorescent di-C4-, otherFeat=[]-->, belongsTo=parr 581 PtdIns(4)P with DrrA (200 nM) was carried out in the presence, otherFeat=[]-->, belongsTo=parr 582 of 400 nM di-C4-PtdIns(4)P and 200 nM BODIPY?PtdIns(4)P., otherFeat=[]-->, belongsTo=parr 583 Data were fitted to a simple competition model yielding the kon, otherFeat=[]-->, belongsTo=parr 584 and koff values for di-C4-PtdIns(4)P by using the program KinTek, otherFeat=[]-->, belongsTo=parr 585 explorer ( Johnson et al, 2009)., otherFeat=[]-->, belongsTo=parr 586 Accession codes. Protein Data Bank: coordinates for the, otherFeat=[]-->, belongsTo=parr 587 DrrA340?647 crystal structure have been deposited under accession, otherFeat=[]-->, belongsTo=parr 588 code 3N6O., otherFeat=[]-->, belongsTo=parr 589 Supplementary information is available at EMBO reports online, otherFeat=[]-->, belongsTo=parr 590 (http://www.emboreports.org)., otherFeat=[u'a']-->, belongsTo=parr 591 ACKNOWLEDGEMENTS, otherFeat=['U']-->, belongsTo=parrnote 592 C. Herrmann and M. Wehner from the University of Bochum are, otherFeat=[]-->, belongsTo=parrnote 593 acknowledged for help in isothermal titration calorimetry measurements., otherFeat=[]-->, belongsTo=parrnote 594 N. Bleimling is acknowledged for invaluable technical assistance. We, otherFeat=[]-->, belongsTo=parrnote 595 thank the staff of Beamline X10SA at the Paul Scherrer Institute (Villingen,, otherFeat=[]-->, belongsTo=parrnote 596 Switzerland) for access to their facilities and the X-ray communities at the, otherFeat=[]-->, belongsTo=parrnote 597 Max Planck Institute of Molecular Physiology (Dortmund, Germany) and, otherFeat=[]-->, belongsTo=parrnote 598 the Max Planck Institute for Medical Research (Heidelberg, Germany) for, otherFeat=[]-->, belongsTo=parrnote 599 help with data collection., otherFeat=[]-->, belongsTo=parrnote 600 CONFLICT OF INTEREST, otherFeat=['U']-->, belongsTo=parrnote 601 The authors declare that they have no conflict of interest., otherFeat=[]-->, belongsTo=parrnote 602 REFERENCES, otherFeat=['U']-->, belongsTo=parrnote 603 Adams PD et al (2010) PHENIX: a comprehensive Python-based system for, otherFeat=[]-->, belongsTo=parrnote 604 macromolecular structure solution. 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Schoebel et al, otherFeat=[]-->, belongsTo=nota_cab_pie 683 EMBO reports VOL 11 | NO 8 | 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 684 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, otherFeat=[]-->, belongsTo=nota_cab_pie 685 scientific report, otherFeat=[]-->, belongsTo=nota_cab_pie 686 604, otherFeat=[]-->, belongsTo=nota_cab_pie ============================== 0 High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA-->id=0, page=0, size=33, fam=Times, col=#000000, type=title, textLines=2--->[]--->title High-affinity bindin>>>lla pneumophila DrrA 1 Stefan Schoebel, Wulf Blankenfeldt, Roger S. Goody+ &Aymelt Itzen++-->id=1, page=0, size=15, fam=Times, col=#000000, type=title, textLines=1--->[]--->note Stefan Schoebel, Wul>>>ody+ &Aymelt Itzen++ 2 Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, North Rhine-Westphalia, Germany-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->[]--->note Department of Physic>>>-Westphalia, Germany 3 The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum-derived vesicles to Legionella-containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P-binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr The DrrA protein of >>>ndance of the lipid. 4 Keywords: DrrA; Legionella; phosphatidylinositol 4-phosphate; Rab1; SidM-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Keywords: DrrA; Legi>>>hosphate; Rab1; SidM 5 EMBO reports (2010) 11, 598­604. doi:10.1038/embor.2010.97-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr EMBO reports (2010) >>>0.1038/embor.2010.97 6 INTRODUCTION-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->['U']--->title INTRODUCTION>>>INTRODUCTION 7 The pathogen Legionella pneumophila causes Legionnaires' disease by infecting human lung macrophages (Muder et al, 1986). Shortly after uptake, the bacterium establishes the Legionella-containing vacuole (LCV) as an intracellular replicative organelle, from which it injects several proteins through its type IV secretion system into the cytosol of the host cell (Isberg et al, 2009). One of the substrates of the type IV secretion system is DrrA, also known as SidM, a 647-amino-acid protein that is involved in redirecting endoplasmic reticulum (ER)-derived vesicles to the LCV. DrrA consists of three domains, the central domain (amino acids 340­533) having guanine nucleotide exchange factor (GEF) activity towards the GTPase Rab1 (Machner & Isberg, 2007; Schoebel et al, 2009; Suh et al, 2010), a carboxyterminal lipid-binding domain and an amino-terminal domain of unknown function. Immediately after transfer from the Legionella bacterium into the host cytosol, DrrA localizes to the cytosolic surface of the LCV through its C-terminal domain by binding specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P; Brombacher et al, 2009) . Once bound to the LCV, the GEF domain of DrrA mediates redirection of ER-derived vesicles through the recruitment and activation of Rab1 (Derre & Isberg, 2004; Kagan et al, 2004; Schoebel et al, 2009). As Rab proteins, such as Rab1, can exert the function of regulating vesicular transport only when they are attached to their respective target membrane, the correct membrane localization of the Rabactivating GEFs is crucial. Thus, Legionella ensures the correct targeting and activation of Rab1 during infection to the LCV by attaching DrrA through lipid binding to the LCV.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr The pathogen Legione>>> binding to the LCV. 8 The PtdIns(4)P-binding domain of SidM/DrrA (P4M) has been identified recently (Brombacher et al, 2009). To understand the structural basis for PtdIns(4)P recognition, we have determined the crystal structure of P4M. A subsequent quantification revealed an unusually high binding affinity for the P4M­PtdIns(4)P interaction, which has not been observed for other phosphatidylinositol phosphate (PtdInsP)-binding proteins.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr The PtdIns(4)P-bindi>>>P)-binding proteins. 9 RESULTS AND DISCUSSION-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->['U']--->title RESULTS AND DISCUSSI>>>SULTS AND DISCUSSION 10 Structure of DrrA340­647-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->[]--->title Structure of DrrA340>>>cture of DrrA340­647 11 The minimal fragment of P4M is small, consisting of about 100 amino acids, and has no sequence homology to any known lipidbinding domain. As constructs containing only P4M (amino acids 544­647) seemed to reduce PtdIns(4)P binding to some degree (Brombacher et al, 2009), we determined the crystal structure of a fragment of DrrA comprising the GEF domain and P4M (amino acids 340­647) at 2.5 A° resolution (Fig 1A; for data collection, phasing and refinement statistics, see Table 1). As reported previously for the GEF domain (Schoebel et al, 2009; Suh et al, 2010), P4M has a new protein fold with no homologous structures found in the Protein Data Bank when running a search using the Dali server (Holm et al, 2008). P4M consists of approximately 50% of a-helices (six a-helices and one 310-helix) and 50% of ordered loops. The three central helices aPI2, aPI3 and aPI6 are arranged perpendicularly to a-helices aG6­aG8 of the GEF domain. At the tip of these helices, two sulphate ions from the crystallization buffer are found in a positively charged pocket (Fig 1B), surrounded mainly by the aPI1­aPI2 loop and the aPI4­aPI5 loop (Fig 1C). The sulphate ions are spaced by 7.2 A°, in accordance with the distance between the phosphate groups in PtdIns(4)P (Fig 1C, right). This mimicking property of sulphate ions for binding of the phosphate head groups of PtdInsP has been observed in another instance (p47phox-PX; Karathanassis et al, 2002). It is therefore probable that this positively charged pocket constitutes the binding site for the PtdIns(4)P head group (supplementary Fig S1 online). Furthermore, a negatively charged surface patch is observed on the opposite side of the presumed PtdIns(4)P-binding cavity, which is likely to be repelled by the negatively charged cytosolic surface of intracellular membranes (Fig 1B). This effect will presumably help to orient the protein during the LCV-binding process such that the supposed PtdIns(4)Pbinding pocket faces towards intracellular PtdIns(4)P-containing membranes (Weber et al, 2006) .-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr The minimal fragment>>>Weber et al, 2006) . 12 Received 20 February 2010; revised 9 June 2010; accepted 9 June 2010; published online 9 July 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Received 20 February>>>d online 9 July 2010 13 +Corresponding author. Tel: þ 49 231 1332300; Fax: þ 49 231 1332399; E-mail: roger.goody@mpi-dortmund.mpg.de ++Corresponding author. Tel: þ 49 231 1332305; Fax: þ 49 231 1332399; E-mail: aymelt.itzen@mpi-dortmund.mpg.de-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr +Corresponding autho>>>@mpi-dortmund.mpg.de 14 Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund, North Rhine-Westphalia 44227, Germany-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Department of Physic>>>halia 44227, Germany 15 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 16 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 17 scientificreport-->id=9, page=0, size=29, fam=Times, col=#dedede, type=title, textLines=1--->[]--->note scientificreport>>>scientificreport 18 scientific report-->id=10, page=0, size=72, fam=Times, col=#008c44, type=title, textLines=1--->[]--->note scientific report>>>scientific report 19 598-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 598>>>598 20 The interactions between the P4M and GEF domains are mainly polar (supplementary Fig S2 online), which leads to a defined relative orientation that is probably also stable in solution or when DrrA is bound at the membrane, as is corroborated by the-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr The interactions bet>>> corroborated by the 21 G4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G4>>>G4 22 G2-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G2>>>G2 23 G1-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G1>>>G1 24 G3-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G3>>>G3 25 G6-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G6>>>G6 26 N-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title N>>>N 27 C PI6-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr C PI6>>>C PI6 28 G5-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G5>>>G5 29 G8-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G8>>>G8 30 G7-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr G7>>>G7 31 Pl2-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr Pl2>>>Pl2 32 Pl4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr Pl4>>>Pl4 33 Pl5-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr Pl5>>>Pl5 34 310-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr 310>>>310 35 Pl1-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr Pl1>>>Pl1 36 SO2­ #14-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr SO2­ #14>>>SO2­ #14 37 120°-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 120°>>>120° 38 Tyr 532-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Tyr 532>>>Tyr 532 39 Asp 565-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Asp 565>>>Asp 565 40 Lys 568-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Lys 568>>>Lys 568 41 Ser 620-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Ser 620>>>Ser 620 42 Ser 621-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Ser 621>>>Ser 621 43 Gln 608-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Gln 608>>>Gln 608 44 Thr 612-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Thr 612>>>Thr 612 45 Arg 541-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Arg 541>>>Arg 541 46 ­10 kBT-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­10 kBT>>>­10 kBT 47 10 kBT-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 10 kBT>>>10 kBT 48 SO2­ #2-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr SO2­ #2>>>SO2­ #2 49 4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr 4>>>4 50 Pl3-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr Pl3>>>Pl3 51 His 543-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr His 543>>>His 543 52 SO2­ #24-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr SO2­ #24>>>SO2­ #24 53 SO2­ #14-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr SO2­ #14>>>SO2­ #14 54 ­2O4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr ­2O4>>>­2O4 55 OH-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title OH>>>OH 56 7.2 Å-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title 7.2 Å>>>7.2 Å 57 Ser 621-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Ser 621>>>Ser 621 58 Ser 620-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Ser 620>>>Ser 620 59 Gln 608-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Gln 608>>>Gln 608 60 Lys 568-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Lys 568>>>Lys 568 61 Tyr 532 Arg 541 His 543-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Tyr 532 Arg 541 His >>> 532 Arg 541 His 543 62 Asp 565-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Asp 565>>>Asp 565 63 Thr 612-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Thr 612>>>Thr 612 64 OH-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title OH>>>OH 65 OH OH-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title OH OH>>>OH OH 66 O-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title O>>>O 67 O-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title O>>>O 68 O-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title O>>>O 69 O-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title O>>>O 70 R-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title R>>>R 71 P-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title P>>>P 72 P-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title P>>>P 73 SO2­ crystal structure of a similar DrrA fragment that was published while this paper was under review (Zhu et al, 2010). This structure is nearly identical to the one described here (supplementary Fig S3A online), despite having been obtained at markedly different pH and from crystals that diffract to only 3.5 A°, whereas our model was refined at 2.5 A°. The higher resolution allowed us to identify the position of a second sulphate ion, which leads to the modelling of the presumed position and orientation of PtdIns(4)P, thereby providing a reasonable model for the mechanism of PtdIns(4)P recognition (supplementary Fig S1 online) and membrane binding (see below). There is no structural similarity between P4M and other PtdInsP-binding domains. However, the Epsin N-terminal homology domain of Epsin is also an a-helical protein, and harbours an amphiphatic a-helix (a0) close to the PtdInsP-binding pocket that is implicated in membrane binding (Ford et al, 2002). This is also seen in our structure (supplementary Fig S3B online), supporting the idea that a-helix aPI5 could be involved in membrane binding.-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr SO2­ crystal structu>>>in membrane binding. 74 4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr 4>>>4 75 SO2­-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr SO2­>>>SO2­ 76 4-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr 4>>>4 77 A-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr A>>>A 78 B-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->['U']--->title B>>>B 79 C-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr C>>>C 80 Fig 1 | DrrA reveals a new fold for phosphatidylinositol-4-phosphate binding. (A) Representation of the crystal structure of DrrA340­647. The GEF domain is shown in light blue, P4M in the spectrum from blue to red. Two sulphate ions (SO42À #1 and SO42À #2) found in the crystal structure indicate the presumed PtdIns(4)P-binding pocket and are drawn in stick representation. (B) Surface representations of P4M coloured by its electrostatic potential with two sulphate ions (stick representation) occupying the PtdIns(4)P-binding pocket. (C) Left: polar contacts between sulphate ions (sticks), the sulphate surrounding water molecules (red spheres) and selected residues of P4M. Right: schematic depiction of the polar contacts between DrrA and sulphate ions. The PtdIns(4)P head group is depicted schematically below to illustrate the spacing between 1- and 4-phosphates (sulphates are numbered according to Fig 1A). GEF, guanine nucleotide exchange factor; P4M, PtdIns(4)P-binding domain of SidM/DrrA; PtdIns(4)P, phosphatidylinositol 4-phosphate.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->capfig Fig 1 | DrrA reveals>>>nositol 4-phosphate. 81 Molecular basis of PtdIns(4)P binding by DrrA-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note Molecular basis of P>>>(4)P binding by DrrA 82 S. Schoebel et al-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note S. Schoebel et al>>>S. Schoebel et al 83 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 84 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 85 scientific report-->id=929, page=1, size=36, fam=Times, col=#008c44, type=title, textLines=6--->[]--->note scientific report>>>scientific report 86 59 9-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 59 9>>>59 9 87 Interaction of DrrA340­647 with PtdIns(4)P-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->[]--->title Interaction of DrrA3>>>­647 with PtdIns(4)P 88 To determine the affinity between DrrA and PtdIns(4)P, we performed isothermal titration calorimetry measurements with a water-soluble analogue of PtdIns(4)P, namely, di-C4-PtdIns(4)P. The experiment revealed an unexpectedly high affinity of DrrA towards di-C4-PtdIns(4)P, with a dissociation equilibrium constant (KD) of approximately 30 nM (Fig 2A). In further experiments, it was possible to determine the association and dissociation pocket of P4M containing sulphates is indeed the binding cavity for PtdIns(4)P.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr To determine the aff>>>vity for PtdIns(4)P. 89 rate constants (kon and koff, respectively) of DrrA340­647 by-->id=7, page=0, size=6, fam=Times, col=#000000, type=note, textLines=3--->[]--->note rate constants (kon >>>y) of DrrA340­647 by 90 using a fluorescent analogue of di-C4-PtdIns(4)P labelled with boron-dipyrromethene (BODIPY; BODIPY­PtdIns(4)P), exploiting the change in fluorescence polarization on binding. Fitting the association traces to single exponentials (Fig 2B) and plotting the observed pseudo-first order rate constants against the DrrA concentration resulted in kon ¼ 3.2 Â 106 MÀ1 sÀ1 (Fig 2C). In a separate experiment, the displacement of BODIPY­PtdIns(4)P from DrrA with non-fluorescent di-C4-PtdIns(4)P resulted in a slow dissociation rate constant (koff) of 0.016 sÀ1 (Fig 2C, inset). Together, the KD value for the DrrA­(BODIPY­PtdIns(4)P) interaction is calculated to be 5 nM.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr using a fluorescent >>>lculated to be 5 nM. 91 As it is possible that the high binding affinity between DrrA and BODIPY­PtdIns(4)P is in part due to the presence of the fluorescence group, we performed competition experiments with unlabelled di-C4-PtdIns(4)P (Fig 2D). The displacement of 200 nM BODIPY­PtdIns(4)P from a mixture with 200 nM DrrA340­647 was monitored by the addition of 400 nM di-C4-PtdIns(4)P and was fitted to a simple competition model. From the end point of the reaction, the dissociation constant for di-C4-PtdIns(4)P was determined to be KD ¼ 18.2 nM. Kinetic constants were calculated from the time dependence of the change in fluorescence polarization (kon ¼ 4.1 Â 106 MÀ1 sÀ1, koff ¼ 0.079 sÀ1). Thus, the absence of the fluorescence reporter group resulted in a fourfold weaker di-C4-PtdIns(4)P binding compared with BODIPY­ PtdIns(4)P. Nevertheless, to the best of our knowledge, the affinity of P4M-PtdIns(4)P is at least an order of magnitude higher than that of most PtdInsP­protein interactions reported so far. The strongest interaction with a PtdInsP known to date is between the PH domains of oxysterol-binding protein and PtdIns(4)P and has a KD value of 40­100 nM (Stahelin et al, 2007) .-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr As it is possible th>>>helin et al, 2007) . 92 Owing to the close proximity of the P4M and GEF domains of DrrA in the crystal structure, we investigated whether Rab1b can modulate the affinity of DrrA towards PtdIns(4)P. For this purpose, we measured the time-dependent displacement of BODIPY­ PtdIns(4)P from DrrA340­647 with unlabelled PtdIns(4)P in the presence of Rab1b:GDP and Rab1b:GTP (supplementary Fig S4 online). The rate of dissociation of BODIPY­PtdIns(4)P from the complex was unchanged, regardless of the presence of Rab1b: GDP, Rab1b:GTP or the nucleotide-free Rab1b:DrrA340­647 complex, indicating that Rab1b has no modulatory role on PtdIns(4)P affinity for P4M.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Owing to the close p>>>)P affinity for P4M. 93 Using the fluorescent binding assay, we also investigated the relevance of the positively charged pocket of P4M of DrrA for PtdIns(4)P binding. As the sulphate ions could potentially mimic the position of the 4-phosphate head group of PtdIns(4)P, we mutated the sulphate-interacting residue Lys 568 to alanine (Fig 1C). Melting point analysis by circular dichroism showed no effect on protein stability (supplementary Fig S5 online), but the mutation completely abolished the DrrA­(BODIPY­PtdIns(4)P) interaction (Fig 2E). This result confirms that the positively charged-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Using the fluorescen>>>e positively charged 94 Table 1 | Data collection, phasing and refinement statistics-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Table 1 | Data colle>>>efinement statistics 95 DrrA340­647-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr DrrA340­647>>>DrrA340­647 96 DrrA340­647 SeMet*-->id=7, page=0, size=6, fam=Times, col=#000000, type=note, textLines=3--->[]--->note DrrA340­647 SeMet*>>>DrrA340­647 SeMet* 97 Data collectionz Space group P212121 P212121-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Data collectionz Spa>>>roup P212121 P212121 98 Cell dimensions a, b, c (A° ) 74.9, 75.4, 131.0 74.1, 75.3, 131.0-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Cell dimensions a, b>>>.0 74.1, 75.3, 131.0 99 a, b, g ( 1)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr a, b, g ( 1)>>>a, b, g ( 1) 100 90, 90, 90-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 90, 90, 90>>>90, 90, 90 101 90, 90, 90-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 90, 90, 90>>>90, 90, 90 102 Resolution (A° )y-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Resolution (A° )y>>>Resolution (A° )y 103 20­2.5 (2.6­2.5)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 20­2.5 (2.6­2.5)>>>20­2.5 (2.6­2.5) 104 20­2.7 (2.8­2.7)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 20­2.7 (2.8­2.7)>>>20­2.7 (2.8­2.7) 105 Rmean-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Rmean>>>Rmean 106 11.1 (41.2)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 11.1 (41.2)>>>11.1 (41.2) 107 15.6 (46.2)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 15.6 (46.2)>>>15.6 (46.2) 108 I/sI-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr I/sI>>>I/sI 109 15.8 (4.2)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 15.8 (4.2)>>>15.8 (4.2) 110 10.6 (4.0)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 10.6 (4.0)>>>10.6 (4.0) 111 Completeness (%)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Completeness (%)>>>Completeness (%) 112 100 (100)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 100 (100)>>>100 (100) 113 100 (100)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 100 (100)>>>100 (100) 114 Redundancy-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Redundancy>>>Redundancy 115 8.1 (8.2)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 8.1 (8.2)>>>8.1 (8.2) 116 7.3 (7.1)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 7.3 (7.1)>>>7.3 (7.1) 117 Refinement-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Refinement>>>Refinement 118 Resolution (A° )-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Resolution (A° )>>>Resolution (A° ) 119 2.5-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 2.5>>>2.5 120 No. of reflections-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr No. of reflections>>>No. of reflections 121 26,298-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 26,298>>>26,298 122 Rwork/Rfree-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Rwork/Rfree>>>Rwork/Rfree 123 18.9/26.2-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 18.9/26.2>>>18.9/26.2 124 No. of atoms-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr No. of atoms>>>No. of atoms 125 Protein-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Protein>>>Protein 126 4,752-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 4,752>>>4,752 127 Ligand/ion-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Ligand/ion>>>Ligand/ion 128 40-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 40>>>40 129 Water-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Water>>>Water 130 174-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 174>>>174 131 B-factors-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr B-factors>>>B-factors 132 Protein-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Protein>>>Protein 133 35-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 35>>>35 134 Ligand/ion-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Ligand/ion>>>Ligand/ion 135 42-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 42>>>42 136 Water-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Water>>>Water 137 40-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 40>>>40 138 r.m.s.d.J-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr r.m.s.d.J>>>r.m.s.d.J 139 Bond lengths (A° )-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Bond lengths (A° )>>>Bond lengths (A° ) 140 0.019-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 0.019>>>0.019 141 Bond angles ( 1)-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr Bond angles ( 1)>>>Bond angles ( 1) 142 1.658-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr 1.658>>>1.658 143 *Data collection statistics for single-wavelength anomalous dispersion data refer to unmerged Friedel pairs; zdata sets were collected from a single crystal; yvalues in parentheses refer to the highest resolution shell; Jr.m.s.d. values.-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr *Data collection sta>>>l; Jr.m.s.d. values. 144 Molecular basis of PtdIns(4)P binding by DrrA-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note Molecular basis of P>>>(4)P binding by DrrA 145 S. Schoebel et al-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note S. Schoebel et al>>>S. Schoebel et al 146 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 147 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 148 scientific report-->id=929, page=1, size=36, fam=Times, col=#008c44, type=title, textLines=6--->[]--->note scientific report>>>scientific report 149 600-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 600>>>600 150 Biological relevance of the high affinity of PtdIns(4)P-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->[]--->title Biological relevance>>>finity of PtdIns(4)P 151 To recruit Rab1, DrrA must associate with LCV after it has been released into the cytosol of the host cell. PtdIns(4)P is found mainly in the Golgi and to a lesser extent in the plasma membrane (di Paolo & de Camilli, 2006), the latter being the primary origin of LCV. Owing to the presumably low abundance of PtdIns(4)P in the LCV immediately after Legionella phagocytosis and because of possible competition with other PtdIns(4)P-binding proteins, DrrA seems to have evolved to bind to PtdIns(4)P with exceptionally high affinity. DrrA is released from the LCV in the course of the Legionella infectious cycle, so that membrane binding is only transient (Ingmundson et al, 2007) . As binding towards PtdIns(4)P is exceptionally strong, release from the membrane most probably occurs as a result of a reduction of the amount of PtdIns(4)P in the course of the maturation of LCVs. Despite the high affinity, the The structure of DrrA340­647, comprising the GEF domain and the P4M, allows us to propose a structural model for DrrA membrane binding and Rab1 membrane anchoring (Fig 3). DrrA presumably binds to PtdIns(4)P-containing membranes in a manner that orients the GEF domain away from the membrane surface. In this model, P4M can potentially interact with a negatively charged membrane through an arginine residue of the aPI1­aPI2 loop (Arg 544) and through hydrophobic interactions of two leucines of the a-helix aPI5 (Leu 610, Leu 614) with the lipid bilayer. The GEF domain extends into the cytosol and is able to recruit Rab1 from a pool of cytosolic Rab1:GDI (GDP dissociation inhibitor) complexes. Binding of Rab1 and the concomitant nucleotide exchange from GDP to GTP are necessary for effective GDP dissociation inhibitor displacement (Schoebel et al, 2009). In our model, the binding of Rab1 to membrane-bound DrrA would orient the GTPase with the nucleotide-binding pocket facing the cytosol, thus allowing direct access for GTP to the Rab1:DrrA complex. In addition, the C-terminus of Rab1 will be positioned in close proximity to the membrane and could thus facilitate the incorporation of the C-terminal geranylgeranyl moieties of Rab1 into the lipid bilayer. Thus, the P4M of DrrA achieves three goals simultaneously: first, it binds specifically and tightly to PtdIns(4)P and hence localizes DrrA to the LCV. Second, it orients the GEF domain of DrrA in a manner that promotes undisturbed nucleotide exchange on Rab1. Third, P4M brings the prenylated C-terminus of Rab1 in close proximity to the membrane, thereby supporting the insertion of geranylgeranyl moieties and leading to stable attachment of Rab1 to the LCV for subsequent recruitment of ER-derived vesicles.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr To recruit Rab1, Drr>>>ER-derived vesicles. 152 DrrA:PtdIns(4)P complex is moderately dynamic (koff ¼ 0.079 sÀ1;-->id=7, page=0, size=6, fam=Times, col=#000000, type=note, textLines=3--->[]--->note DrrA:PtdIns(4)P comp>>>c (koff ¼ 0.079 sÀ1; 153 that is, half-life ¼ ln2/koff ¼ 8.7 s) and would therefore allow for PtdIns(4)P modifications on a physiologically relevant time scale. The high specificity of DrrA for PtdIns(4)P (Brombacher et al, 2009), together with its exceptionally high binding affinity, makes P4M a potentially valuable molecular tool for the specific and sensitive labelling of intracellular membranes for the presence of PtdIns(4)P. As proof of principle, it was shown that the PH domains of oxysterol-binding protein and four-phosphate adaptor protein 1 can be used to monitor the cellular activity and spatial distribution of the PtdIns(4)P-generating enzyme PtdIns(4) kinase (Balla et al, 2005). However, the tighter interaction of DrrA with PtdIns(4)P makes the protein potentially even better suited to this purpose.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr that is, half-life ¼>>>ted to this purpose. 154 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 155 020-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 020>>>020 156 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 157 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 158 50 100 150 Time (ms) Time (s)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 50 100 150 Time (ms)>>>0 Time (ms) Time (s) 159 Time (s)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Time (s)>>>Time (s) 160 DrrA340­647 K568A-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr DrrA340­647 K568A>>>DrrA340­647 K568A 161 DrrA 340­647-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr DrrA 340­647>>>DrrA 340­647 162 DrrA340­647 (M)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr DrrA340­647 (M)>>>DrrA340­647 (M) 163 DrrAfl-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr DrrAfl>>>DrrAfl 164 200-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 200>>>200 165 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 166 0.5-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0.5>>>0.5 167 1.0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 1.0>>>1.0 168 0.5-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0.5>>>0.5 169 N = 1.3-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title N = 1.3>>>N = 1.3 170 KD = 30 nM-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr KD = 30 nM>>>KD = 30 nM 171 H = 29,240 cal mol­1 S = ­64 cal mol­1 K­1-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr H = 29,240 cal mol­1>>> = ­64 cal mol­1 K­1 172 1.0 1.5 Molar ratio-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 1.0 1.5 Molar ratio>>>1.0 1.5 Molar ratio 173 2.0 2.5-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 2.0 2.5>>>2.0 2.5 174 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 175 100 200 300 400 500-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 100 200 300 400 500>>>100 200 300 400 500 176 6.5 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 6.5 M>>>6.5 M 177 3.25 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 3.25 M>>>3.25 M 178 1.63 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 1.63 M>>>1.63 M 179 0.81 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0.81 M>>>0.81 M 180 0.40 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0.40 M>>>0.40 M 181 13 M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 13 M>>>13 M 182 40-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 40>>>40 183 60-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 60>>>60 184 Time (min)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Time (min)>>>Time (min) 185 cal-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr cal>>>cal 186 s-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr s>>>s 187 ­1-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr ­1>>>­1 188 kcal-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr kcal>>>kcal 189 per-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr per>>>per 190 mole-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr mole>>>mole 191 of-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr of>>>of 192 injectant-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr injectant>>>injectant 193 FP-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title FP>>>FP 194 (mP)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr (mP)>>>(mP) 195 FP-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title FP>>>FP 196 (mP)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr (mP)>>>(mP) 197 FP-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title FP>>>FP 198 (mP)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr (mP)>>>(mP) 199 ­1-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­1>>>­1 200 ­2-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­2>>>­2 201 ­10-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­10>>>­10 202 ­20-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­20>>>­20 203 ­30-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­30>>>­30 204 200-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 200>>>200 205 150-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 150>>>150 206 100-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 100>>>100 207 50-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 50>>>50 208 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 209 200-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 200>>>200 210 100-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 100>>>100 211 120-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 120>>>120 212 140-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 140>>>140 213 160-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 160>>>160 214 150-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 150>>>150 215 100-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 100>>>100 216 50-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 50>>>50 217 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 218 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 219 ­3-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ­3>>>­3 220 A-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr A>>>A 221 D-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->['U']--->title D>>>D 222 E-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->['U']--->title E>>>E 223 B-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->['U']--->title B>>>B 224 Time (s)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Time (s)>>>Time (s) 225 k obs-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr k obs>>>k obs 226 (s-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr (s>>>(s 227 ­1 )-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr ­1 )>>>­1 ) 228 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 229 5 10 15-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 5 10 15>>>5 10 15 230 500-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 500>>>500 231 250-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 250>>>250 232 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 233 FP-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title FP>>>FP 234 (mP)-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr (mP)>>>(mP) 235 40-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 40>>>40 236 30-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 30>>>30 237 20-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 20>>>20 238 10-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 10>>>10 239 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 240 150-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 150>>>150 241 100-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 100>>>100 242 0-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr 0>>>0 243 C-->id=927, page=1, size=10, fam=Times, col=#010202, type=parrnote, textLines=66--->[]--->parr C>>>C 244 Fig 2 | DrrA binds phosphatidylinositol 4-phosphate selectively with unexpectedly high affinity. (A) Isothermal titration calorimetry experiment of-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr Fig 2 | DrrA binds p>>>imetry experiment of 245 50 mM di-C4-PtdIns(4)P titrated into 5 mM DrrA340­647.(B) Individual stopped-flow time traces of the association between 166 nM BODIPY­PtdIns(4)P-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr 50 mM di-C4-PtdIns(4>>>nM BODIPY­PtdIns(4)P 246 and indicated concentrations of DrrA340­647 monitored by the change in fluorescence polarization. (C) Linear fit of the observed DrrA340­647­BODIPY­-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr and indicated concen>>> DrrA340­647­BODIPY­ 247 PtdIns(4)P association rates against DrrA340­647 concentration. Inset: BODIPY­PtdIns(4)P displacement from a DrrA340­647:BODIPY­PtdIns(4)P complex (0.4 mM) by 5 mM di-C4-PtdIns(4)P. (D) Stopped-flow competition binding experiment (red line) using 200 nM DrrA340­647, 200 nM BODIPY­PtdIns(4)P and 400 nM di-C4-PtdIns(4)P. The data were fitted to a simple competition model for the determination of KD, kon and koff-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr PtdIns(4)P associati>>> of KD, kon and koff 248 of di-C4-PtdIns(4)P to DrrA340­647.(E) Association of 166 nM BODIPY­PtdIns(4)P with 6.5 mM DrrAfl, DrrA340­647 or DrrA340­647 K568A.-->id=893, page=1, size=5, fam=Times, col=#677671, type=parrnote, textLines=39--->[]--->parr of di-C4-PtdIns(4)P >>>r DrrA340­647 K568A. 249 BODIPY, boron-dipyrromethene; FP, fluorescence polarization; PtdIns(4)P, phosphatidylinositol 4-phosphate.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr BODIPY, boron-dipyrr>>>nositol 4-phosphate. 250 Molecular basis of PtdIns(4)P binding by DrrA-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note Molecular basis of P>>>(4)P binding by DrrA 251 S. Schoebel et al-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note S. Schoebel et al>>>S. Schoebel et al 252 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 253 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 254 scientific report-->id=929, page=1, size=36, fam=Times, col=#008c44, type=title, textLines=6--->[]--->note scientific report>>>scientific report 255 60 1-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 60 1>>>60 1 256 METHODS-->id=3, page=0, size=12, fam=Times, col=#000000, type=title, textLines=7--->['U']--->title METHODS>>>METHODS 257 Materials. Di-C4-PtdIns(4)P (di-C4-PtdIns(4)P, P-4004) and BODIPY tetramethylrhodamine (TMR) PtdIns(4)P (BODIPY­PtdIns(4)P, C-04M6) were obtained from Echelon.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Materials. Di-C4-Ptd>>>tained from Echelon. 258 Purification of Legionella DrrA340­647 and mutants. DrrA340­647 was subcloned into a modified pET19 vector (Novagen) that contained an N-terminal hexa-histidine tag and a tobacco etch virus (TEV) protease cleavage sequence. The site-specific mutant DrrA340­647 K568A was created by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Proteins were expressed in Escherichia coli (BL21-CodonPlus(DE3)-RIL) at 37 1C after induction with 1 mM isopropyl-b-dithiogalactopyranoside. Selenomethionine-labelled DrrA340­647 was expressed using the methionine biosynthesis inhibition method (van Duyne et al, 1993). After bacterial cell lysis, the supernatant was applied to nickel­nitrilotriacetic acid (Ni­NTA) chromatography and proteins were eluted with a linear Imidazole gradient (5­500 mM) in buffer A (20 mM HEPES (pH 8.0), 50 mM NaCl and 1 mM-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Purification of Legi>>> 50 mM NaCl and 1 mM 259 DrrA-GEF-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr DrrA-GEF>>>DrrA-GEF 260 Nucleotide binding site b-mercaptoethanol). Fractions containing DrrA340­647 or the mutant protein were pooled and digested with His10-tagged TEV protease while dialysing against buffer A to remove imidazole. Uncleaved protein and TEV protease were removed by passing over the Ni­NTA column again, and the concentrated sample was subjected to size exclusion chromatography (Superdex75 16/60, GE Healthcare) in buffer containing 20 mM HEPES (pH 8.0), 50 mM NaCl and 2 mM dithiothreitol. The protein was concentrated to 20 mg/ml and flash-frozen in liquid nitrogen. Rab1b purification has been described previously (Schoebel et al, 2009) .-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Nucleotide binding s>>>oebel et al, 2009) . 261 Rab1-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Rab1>>>Rab1 262 N-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->['U']--->title N>>>N 263 Prenyl moieties-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Prenyl moieties>>>Prenyl moieties 264 ?-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr ?>>>? 265 DrrA amino-terminal domain-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr DrrA amino-terminal >>>mino-terminal domain 266 C-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr C>>>C 267 DrrA-P4M-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr DrrA-P4M>>>DrrA-P4M 268 Ptdlns(4)P-->id=6, page=0, size=7, fam=Times, col=#000000, type=note, textLines=1--->[]--->note Ptdlns(4)P>>>Ptdlns(4)P 269 Membrane-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->parr Membrane>>>Membrane 270 Fig 3 | Structural model of DrrA membrane binding and simultaneous Rab1 interaction. The model demonstrates a possible orientation of P4M towards-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr Fig 3 | Structural m>>>ation of P4M towards 271 a membrane and the implications for Rab1 binding to the DrrA­GEF domain. In this view, P4M orients the preceding GEF domain such that the GDP/GTP-binding pocket of Rab1 points to the cytosol. Concomitantly, the carboxyl terminus of the GTPase is positioned in close proximity to the membrane, thereby presumably facilitating the incorporation of the prenyl moieties into the LCV membrane. The position of Rab1 on DrrA340­647 was modelled by superimposing DrrA340­647 with the Rab13­174:DrrA340­533 complex structure (Schoebel et al, 2009). Green, DrrA-P4M; light blue, DrrA-GEF domain; grey sphere, DrrA amino-terminal domain; orange, Rab1; ball and sticks, PtdIns(4)P model; dashed circle, GDP/GTP bindings site of Rab1; red bars, schematic for Rab1 C-terminal prenyl moieties. GEF, guanine nucleotide exchange factor; LCV, Legionella-containing vacuole; P4M, PtdIns(4)P-binding domain of SidM/DrrA; PtdIns(4)P, phosphatidylinositol 4-phosphate.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr a membrane and the i>>>nositol 4-phosphate. 272 Molecular basis of PtdIns(4)P binding by DrrA-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note Molecular basis of P>>>(4)P binding by DrrA 273 S. Schoebel et al-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->note S. Schoebel et al>>>S. Schoebel et al 274 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 275 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 276 scientific report-->id=929, page=1, size=36, fam=Times, col=#008c44, type=title, textLines=6--->[]--->note scientific report>>>scientific report 277 602-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 602>>>602 278 Crystallization and structure determination of DrrA340­647. Native and selenomethionine-labelled crystals of DrrA340­647 were obtained by the hanging-drop vapour diffusion method by mixing 1 ml of protein (20 mg/ml) in gel filtration buffer with 1 mlof a reservoir solution containing 12% (w/v) polyethylene glycol 4000, 20% (v/v) glycerol, 0.16 M ammonium sulphate and 0.1 M sodium acetate at pH 4.8. Diffraction data were collected on beamline X10SA of the Swiss Light Source and processed with XDS (Kabsch, 1993 ). Selenium atoms were located and a model was obtained with autoSHARP (Vonrhein et al, 2007) in MIR(AS) mode. Structure refinement was completed by using phenix.refine (Adams et al, 2007) and manual rebuilding in Coot (Emsley & Cowtan, 2004 ) with alternating rounds of refinement in REFMAC5 (Murshudov et al, 1997 ). The protein crystallizes in space group P212121 with two independent copies of DrrA340­647 in the asymmetric unit. No electron density is visible for C-terminal residues 639­647. Molecular presentations were prepared with PyMOL (Delano, 2002) .-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Crystallization and >>>OL (Delano, 2002) . 279 Isothermal titration calorimetry of DrrA­PtdIns(4)P interaction. Di-C4-PtdIns(4)P (50 mM) in running buffer (20 mM HEPES (pH 8.0), 50 mM NaCl, 1 mM tris (2-carboxyethyl) phosphine) was titrated automatically into a 5 mMsolution of DrrA340­647 dialysed in the same buffer, using an iTC200 Micro Calorimeter (MicroCal). Titrations were performed at 25 1C, with an injection volume of 6 ml at time intervals of 5 min to ensure that the titration peak returned to baseline. Data were collected using iTC200 control software and analysed with Origin (Version 7.0, MicroCal). Data were corrected for heat of dilution of Di-C4-PtdIns(4)P into buffer, which was determined in a separate titration.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Isothermal titration>>> separate titration. 280 Kinetic analysis of PtdIns(4)P interaction with DrrA. All measurements were carried out at 25 1C in buffer that contained 20 mM HEPES (pH 8.0), 50 mM NaCl and 2 mM dithiothreitol. Fast kinetic measurements were performed with a stoppedflow apparatus (Applied Photophysics). For BODIPY TMR PtdIns(4)P binding or release, time-dependent changes in fluorescence polarization were monitored with a 570nm cutoff filter in the stopped-flow machine, excited at 546 nm. The competition experiment to determine the affinity of non-fluorescent di-C4PtdIns(4)P with DrrA (200 nM) was carried out in the presence of 400 nM di-C4-PtdIns(4)P and 200 nM BODIPY­PtdIns(4)P. Data were fitted to a simple competition model yielding the kon and koff values for di-C4-PtdIns(4)P by using the program KinTek explorer ( Johnson et al, 2009).-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Kinetic analysis of >>>ohnson et al, 2009). 281 Accession codes. Protein Data Bank: coordinates for the DrrA340­647 crystal structure have been deposited under accession code 3N6O.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Accession codes. Pro>>>accession code 3N6O. 282 Supplementary information is available at EMBO reports online-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[]--->parr Supplementary inform>>> EMBO reports online 283 (http://www.emboreports.org).-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=309--->[u'a']--->parr (http://www.emborepo>>>ww.emboreports.org). 284 ACKNOWLEDGEMENTS-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->['U']--->title ACKNOWLEDGEMENTS>>>ACKNOWLEDGEMENTS 285 C. Herrmann and M. Wehner from the University of Bochum are-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr C. Herrmann and M. W>>>ersity of Bochum are 286 acknowledged for help in isothermal titration calorimetry measurements. N. Bleimling is acknowledged for invaluable technical assistance. We-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr acknowledged for hel>>>nical assistance. We 287 thank the staff of Beamline X10SA at the Paul Scherrer Institute (Villingen, Switzerland) for access to their facilities and the X-ray communities at the Max Planck Institute of Molecular Physiology (Dortmund, Germany) and the Max Planck Institute for Medical Research (Heidelberg, Germany) for help with data collection.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr thank the staff of B>>>ith data collection. 288 CONFLICT OF INTEREST-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->['U']--->title CONFLICT OF INTEREST>>>CONFLICT OF INTEREST 289 The authors declare that they have no conflict of interest.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->[]--->parr The authors declare >>>onflict of interest. 290 REFERENCES-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=121--->['U']--->title REFERENCES>>>REFERENCES 291 Adams PD et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. 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Schoebel et al 328 EMBO reports VOL 11 | NO 8 | 2010-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note EMBO reports VOL 11 >>>VOL 11 | NO 8 | 2010 329 &2010 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note &2010 EUROPEAN MOLEC>>>BIOLOGY ORGANIZATION 330 scientific report-->id=929, page=1, size=36, fam=Times, col=#008c44, type=title, textLines=6--->[]--->note scientific report>>>scientific report 331 604-->id=5, page=0, size=8, fam=Times, col=#000000, type=parrnote, textLines=196--->[]--->note 604>>>604