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• W2010236991 abstract "The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser29 to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11- and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway. The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser29 to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11- and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway. Rab proteins form a large family of small GTP-binding proteins with a wide range of functions in the coordination of vesiculo-tubular traffic (1Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar), which include directional tethering, docking and fusion of vesicles to target membranes (2Chavrier P. Goud B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (422) Google Scholar), vesicle motility (3Hammer 3rd, J.A. Wu X.S. Curr. Opin. Cell Biol. 2002; 14: 69-75Crossref PubMed Scopus (124) Google Scholar), and cargo recruitment (4Smythe E. Mol. Cell. 2002; 9: 205-206Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This diversity of functions is essential for determining transport specificity and organelles identity, and is accounted for by the multitude of Rab proteins (at least 60 members in humans) (5Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2001; 313: 889-901Crossref PubMed Scopus (613) Google Scholar). With an average sequence identity little above 30%, they form a heterogeneous family that can be classified into subfamilies based on both functional and sequence relationships (5Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2001; 313: 889-901Crossref PubMed Scopus (613) Google Scholar, 6Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (374) Google Scholar). The cellular functions of Rab proteins are implemented by their ability to couple a structural GDP/GTP switch, common to all small GTP-binding of the Ras superfamily, to a membrane/cytosol switch that depends on lipid modifications attached to their C terminus (7Pereira-Leal J.B. Hume A.N. Seabra M.C. FEBS Lett. 2001; 498: 197-200Crossref PubMed Scopus (147) Google Scholar). In the simplest scheme, GDP-bound Rab proteins are maintained in the cytosol by RabGDIs, 1The abbreviations used are: GDI, guanine dissociation inhibitor; Tf, transferrin; TfR, transferrin receptor; Rab11wt, wild type human Rab11a; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid; GTPγS, guanosine 5′-O-(thiotriphosphate); GppNHp, guanyl-5′-imidodiphosphate. which deliver them to donor membranes where they are activated by guanine nucleotide exchange factors. GTP-bound Rabs then bind to effectors until they are turned off by GTPase activating proteins and recycled back to the cytosol by RabGDIs. This basic model, in which all events happen in sequence, is increasingly challenged by recent studies. For instance, it has been shown that guanine nucleotide exchange factors and effectors can be associated in complexes (8Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 9Wurmser A.E. Sato T.K. Emr S.D. J. Cell Biol. 2000; 151: 551-562Crossref PubMed Scopus (324) Google Scholar) and act in synergy (10Lippe R. Miaczynska M. Rybin V. Runge A. Zerial M. Mol. Biol. Cell. 2001; 12: 2219-2228Crossref PubMed Scopus (158) Google Scholar), or that GDP/GTP cycles can occur without recycling to the cytosol (11Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.C. Goody R. Zerial M. Nature. 1996; 383: 266-269Crossref PubMed Scopus (267) Google Scholar, 12Calhoun B.C. Lapierre L.A. Chew C.S. Goldenring J.R. Am. J. Physiol. 1998; 275: C163-C170Crossref PubMed Google Scholar). Rab proteins also appear to be multifunctional and have multiple effectors often unrelated to each other (1Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar, 13Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). In apparent contradiction to this diversity, other partners display promiscuity to various Rab proteins, such as Rab Escort Proteins (14Pereira-Leal J.B. Strom M. Godfrey R.F. Seabra M.C. Biochem. Biophys. Res. Commun. 2003; 301: 92-97Crossref PubMed Scopus (19) Google Scholar), RabGDI (15Chen W. Feng Y. Chen D. Wandinger-Ness A. Mol. Biol. Cell. 1998; 9: 3241-3257Crossref PubMed Scopus (319) Google Scholar), or the MSS4 regulator (16Burton J.L. Burns M.E. Gatti E. Augustine G.J. De Camilli P. EMBO J. 1994; 13: 5547-5558Crossref PubMed Scopus (106) Google Scholar, 17Zhu Z. Delprato A. Merithew E. Lambright D.G. Biochemistry. 2001; 40: 15699-15706Crossref PubMed Scopus (9) Google Scholar). This complex implementation addresses the issue of how Rab proteins utilize their cycle to fulfill their cellular functions. This applies in particular to the Rab11 protein whose structural and mutational analysis is reported here. This subfamily comprises the ubiquitous Rab11a and Rab11b and the closely related epithelial Rab25 proteins. The best characterized function of Rab11 is the control of TfR recycling and its routing at the exit from recycling endosomes in the pericentriolar region (18Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1080) Google Scholar, 19Ren M. Xu G. Zeng J. De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Crossref PubMed Scopus (392) Google Scholar, 20Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Crossref PubMed Scopus (316) Google Scholar). The classical mutations of small GTP-binding proteins that affect their nucleotide binding and GTPase properties have been extensively used to investigate these functions. In non-polarized cells, overexpression of the dominant-negative Rab11(S25N) mutant redistributes TfR in thin tubular structures that concentrate in a perinuclear network, whereas wild type Rab11 and the constitutively active Rab11(Q70L) mutant induce the formation of tubulo-vesicular TfR-positive organelles at the cell periphery (20Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Crossref PubMed Scopus (316) Google Scholar, 21Holtta-Vuori M. Tanhuanpaa K. Mobius W. Somerharju P. Ikonen E. Mol. Biol. Cell. 2002; 13: 3107-3122Crossref PubMed Scopus (112) Google Scholar). Rab11 also regulates a set of related endosomal functions such as the mobilization of endosomal membranes for phagocytosis in macrophages (22Cox D. Lee D.J. Dale B.M. Calafat J. Greenberg S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 680-685Crossref PubMed Scopus (187) Google Scholar), the recycling of apical endosomes in Madin-Darby canine kidney cells (23Casanova J.E. Wang X. Kumar R. Bhartur S.G. Navarre J. Woodrum J.E. Altschuler Y. Ray G.S. Goldenring J.R. Mol. Biol. Cell. 1999; 10: 47-61Crossref PubMed Scopus (347) Google Scholar), the targeting of secretory vesicles in gastric parietal cells (12Calhoun B.C. Lapierre L.A. Chew C.S. Goldenring J.R. Am. J. Physiol. 1998; 275: C163-C170Crossref PubMed Google Scholar), the modulation of cellular cholesterol transport and homeostasis (21Holtta-Vuori M. Tanhuanpaa K. Mobius W. Somerharju P. Ikonen E. Mol. Biol. Cell. 2002; 13: 3107-3122Crossref PubMed Scopus (112) Google Scholar), and the sorting of membrane proteins to melanosomes in melanocytes. 2F. Senic-Matuglia, H. de la Salle, D. Fricker, B. Goud, and J. Salamero, submitted for publication. In line with this diversity, Rab11 has various effectors that include Rabphilin-11, a protein with six WD-repeat domains (24Zeng J. Ren M. Gravotta D. De Lemos-Chiarandini C. Lui M. Erdjument-Bromage H. Tempst P. Xu G. Shen T.H. Morimoto T. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2840-2845Crossref PubMed Scopus (70) Google Scholar), the FIP family characterized by a conserved Rab11 binding domain (25Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 26Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 299: 770-779Crossref PubMed Scopus (51) Google Scholar), and the tail of the myosin Vb motor (27Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Provance Jr., D.W. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (345) Google Scholar). Structural and mutagenesis studies provide a means to gain insight into the unique structure-function relationships of Rab proteins. Recent structural studies of the GDP/GTP cycle of Rab proteins have shown that, despite their non-conventional cellular cycle, they do not depart from other small GTP-binding proteins and respond to the alternation of GDP and GTP by conformational changes and disorder-to-order transitions at the so-called switch 1 and switch 2 regions (28Stroupe C. Brunger A.T. J. Mol. Biol. 2000; 304: 585-598Crossref PubMed Scopus (89) Google Scholar, 29Constantinescu A.T. Rak A. Alexandrov K. Esters H. Goody R.S. Scheidig A.J. Structure. 2002; 10: 569-579Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). These studies of their GDP/GTP cycles, together with the structure of the complex of Rab3 with its effector Rabphilin3 (30Ostermeier C. Brunger A.T. Cell. 1999; 96: 363-374Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), the structures of Rab proteins bound to either GDP (31Chattopadhyay D. Langsley G. Carson M. Recacha R. DeLucas L. Smith C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 937-944Crossref PubMed Scopus (26) Google Scholar) or GTP analogues (32Dumas J.J. Zhu Z. Connolly J.L. Lambright D.G. Structure Fold Des. 1999; 7: 413-423Abstract Full Text Full Text PDF Scopus (102) Google Scholar, 33Esters H. Alexandrov K. Constantinescu A.T. Goody R.S. Scheidig A.J. J. Mol. Biol. 2000; 298: 111-121Crossref PubMed Scopus (27) Google Scholar, 34Merithew E. Hatherly S. Dumas J.J. Lawe D.C. Heller-Harrison R. Lambright D.G. J. Biol. Chem. 2001; 276: 13982-13988Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 35Zhu G. Liu J. Terzyan S. Zhai P. Li G. Zhang X.C. J. Biol. Chem. 2003; 278: 2452-2460Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and genome-wide analysis of Rab sequences (5Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2001; 313: 889-901Crossref PubMed Scopus (613) Google Scholar, 6Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (374) Google Scholar), suggest that the structural specificity of Rab subfamilies resides in the combination of the nucleotide-sensitive switch regions with nucleotide-insensitive regions featuring subfamily specific sequences and/or conformations. These latter regions have been termed RabSF1–4 and include the N terminus/β1, α1/switch 1, α3–β5, and α5/C terminus, respectively (6Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (374) Google Scholar). Yet, the implementation of the dual nucleotide/membrane cycle of Rab proteins, and how this cycle is distributed over various effectors, remain open questions. In this study, we report the high resolution crystal structures of inactive GDP-bound and active GTPγS-bound human Rab11a. These structures yield a novel hypothesis as to the coupling between the GDP/GTP and membrane/cytosol cycles, which would involve a membrane-bound Rab11-GDP dimer, and they delineate a novel site of interaction for Rab11 partners. This site was investigated by site-directed mutagenesis and the resulting mutants were assayed for the distribution of endosomal markers and endosomal transferrin recycling. This analysis revealed a novel phenotype distinct from that of previously characterized Rab11 mutants, which provides insights into the recognition of Rab11 by distinct effectors. cDNA Cloning and Protein Expression—Because initial trials to crystallize full-length human Rab11a failed, a truncated form (residues 1 to 173) lacking the hypervariable 43 C-terminal residues was used throughout the crystallographic study (referred to as Rab11 hereafter). In addition, a mutation impairing the spontaneous GTPase, Q70L, was introduced to grow crystals of the active form. Constructs of Rab11 and the Q70L mutant were generated by PCR using previously described pGEM-Rab11 constructs (20Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Crossref PubMed Scopus (316) Google Scholar) as templates and the following oligonucleotides: R11-forward (5′-CGCGGATCCTTATATGTTCTGACAGCA-3′) and R11DeltaTyr173 (5′-TTTCTGCAGTCAGTATATCTCTGTCAGAAT-3′). The PCR products were then cloned as BamHI/PstI inserts in pQE32 vector (Qiagen). His-tagged Rab11 proteins were overexpressed in Escherichia coli, purified by nickel affinity and stored in 50 mm Tris-HCl, pH 8, 100 mm NaCl, 2 mm MgCl2, 0.1% 2-mercaptoethanol. Full-length Ser29 → Phe (S29F), Ala49 → Lys (A49K), and Arg33 → Asp (R33D) Rab11 mutants fused to the enhanced green fluorescent protein (EGFP) were obtained with the QuikChange™ XL site-directed mutagenesis kit according to the manufacturer's instructions using pEGFP-Rab11wt (generous gift of Dr. L. Gordon) as a template and the following oligonucleotides: S29F-forward (5′-AGTAATCTCTTGTTTCGATTTACTCGA-3′), S29F-reverse (5′-TCGAGTAAATCGAAACAAGAGATTACT-3′), A49K-forward (5′-GGAGTAGAGTTTAAAACAAGAAGCATC-3′), A49K-reverse (5′-GATGCTTCTTGTTTTAAACTCTACTCC-3′), R33D-forward (5′-TCTCGATTTACTGATAATGAGTTTAAT-3′), and R33D-reverse (5′-ATTAAACTCATTATCAGTAAATCGAGA-3′). Protein Crystallization—All crystallizations were performed at 20 °C by the vapor diffusion method. Crystals of Rab11-GDP (10 mg/ml) appeared within a few days in 1.6–1.8 m (NH4)2SO4, 3–6% PEG 400, 3–10% 1,3-butanediol, 100 mm Tris-HCl, pH 8–8.5, and were optimized by microseeding. Prior to flash-freezing in liquid ethane, crystals were rapidly soaked in a stabilizing solution containing 33% glycerol as a cryoprotectant. Because no Mg2+ could be located in the electron density map under these conditions, further crystallizations were done with 8 mm MgCl2 in the crystallization solution. Crystals of Rab11(Q70L)-GTPγS (10 mg/ml) were grown in the presence of 5 mm GTPγS. Nucleotide exchange was allowed to proceed spontaneously at high Mg2+ concentration, because EDTA rapidly precipitated the protein. Crystals appeared within 5–10 days in 1.1–1.5 m NaCl, 150–200 mm NaH2PO4, 150–200 mm KH2PO4, 100 mm Na-MES, pH 6.5, and were stabilized in the reservoir solution supplied with 22% xylitol prior to flash-freezing. Data Collection, Structure Determination, and Analysis—All diffraction data sets were collected at 100 K at beamline ID14 at the ESRF synchrotron in Grenoble. Data were processed with DENZO/SCALEPACK (36Otwinowski Z. Minor W. Methods Enzymol. 1994; 276: 307-326Crossref Scopus (38573) Google Scholar). Phases were obtained by molecular replacement with AMoRe (37Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar). Refinements were performed with CNS (38Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) and model building was carried out with TURBO. 3afmb.cnrs-mrs.fr. Sec4-GDP (Protein Data Bank entry 1G16 (28Stroupe C. Brunger A.T. J. Mol. Biol. 2000; 304: 585-598Crossref PubMed Scopus (89) Google Scholar)) with the switch regions removed was found to be the best molecular replacement search model for the Rab11-GDP structure. The asymmetric unit comprises two Rab11-GDP molecules related by a non-crystallographic 2-fold axis. Refined Rab11-GDP was then used as a search model for Rab11(Q70L)-GTPγS. The refined structures of Rab11-GDP and Rab11-GTPγS have no Ramachandran outliers. Crystallographic statistics are summarized in Table I. Coordinates have been deposited with the Protein Data Bank with entry codes 1OIV (Rab11-GDP) and 1OIW (Rab11(Q70L)-GTPγS).Table ICrystallographic statistics Data collection statistics.CrystalRab11a-GDPRab11a-GTPγSResolution limits (Å)30—1.95 (1.98—1.95)30—1.90 (1.97—1.90)Space groupP2121211422Unit cell parameters a (Å)47.473.7 b (Å)69.773.7 c (Å)108.3125.2 α = β = γ (°)9090Reflections Measured233617207365 Unique2610013984Completeness (%)97.2 (97.4)96.5 (81.6)Rsymm (%)aRsymm = 100 × ΣI — 〈I〉/ΣI, I is the observed intensity and 〈I〉 is the average intensity calculated from multiple observation of symmetry related reflections. Rsymm given for I/sigmal ≥ 04.9 (39.3)9.6 (28.8)I/σ26.1 (4.8)22.2 (9.4)Structure refinement Resolution limits (Å)30—1.98 (2.03—1.98)30—2.30 (2.40—2.30) Reflections for Rcryst/Rfree23252/17027369/638 Rcryst/Rfree (%)bRcryst = 100 × Σ|Fo — Fc|/Σhkl|Fo|, where Fo and Fc are observed and calculated amplitudes, respectively. Rfree is an Rcryst calculated using a subset of reflections, chosen randomly, kept constant and omitted from all subsequent structure refinement steps. Rcryst and Rfree given for F/sigma≥021.0/24.6 (23.9/27.2)22.5/23.3 (24.8/32.3) R.m.s.d. bond length (Å)0.0050.016 R.m.s.d. angles (°)1.21.7 Average B-factor (Å2)35.031.0a Rsymm = 100 × ΣI — 〈I〉/ΣI, I is the observed intensity and 〈I〉 is the average intensity calculated from multiple observation of symmetry related reflections. Rsymm given for I/sigmal ≥ 0b Rcryst = 100 × Σ|Fo — Fc|/Σhkl|Fo|, where Fo and Fc are observed and calculated amplitudes, respectively. Rfree is an Rcryst calculated using a subset of reflections, chosen randomly, kept constant and omitted from all subsequent structure refinement steps. Rcryst and Rfree given for F/sigma≥0 Open table in a new tab Multiple structure alignments were performed with TURBO using Rab11(Q70L)-GTPγS as a reference. A cutoff distance of 0.5 Å for including Cα pairs in the refinement of the superposition matrix was found to robustly superimpose conserved secondary structures without artifactually averaging significant structural differences. Figures were generated with Molscript (39Kraulis P.J. J. Appl. Crystallog. 1991; 24: 946-950Crossref Google Scholar) and rendered with Raster3D (40Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). Cell Culture, Transfection, and Immunocytochemistry—HeLa cells were maintained in Dulbecco's modified Eagle's medium as previously described (20Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Crossref PubMed Scopus (316) Google Scholar). Transfections were performed by calcium phosphate precipitation and expression of GFP constructs was allowed for 24 h. For immunofluorescence, cells were fixed with 3% paraformaldehyde in phosphate-buffered saline. After permeabilization markers were visualized by double labeling with primary antibodies followed by incubation with Cy3- and Cy5-conjugated anti-IgG (Molecular Probes). Coverslips were mounted in Mowiol (Hoechst AG). Confocal laser scanning microscopy was performed using a SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). Confocal sections or slice projections were further processed using Adobe Photoshop. The following antibodies were used: sheep anti-TGN46 antibody (Serotec), goat anti-EEA1 (Santa Cruz Biotechnology), monoclonal anti-human TfR mouse antibody DF1513 (Sigma), fluorescein isothiocyanate, Cy3 and Cy5 donkey secondary antibodies (Jackson ImmunoResearch). Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, streptomycin, sodium pyruvate, and glutamine were from Invitrogen. Human transferrin-Alexa 633 was from Molecular Probes. The antibody against Rab11 was purified from a rabbit antiserum raised against full-length recombinant Rab11 as described (20Wilcke M. Johannes L. Galli T. Mayau V. Goud B. Salamero J. J. Cell Biol. 2000; 151: 1207-1220Crossref PubMed Scopus (316) Google Scholar). Transferrin Internalization and Recycling—For steady state internalization of transferrin, cells were incubated for 30 min at 37 °C with Alexa 633-labeled Tf to a final concentration of 5 μg/ml in internalization medium (Dulbecco's modified Eagle's medium, 10 mm Hepes, pH 7.4, 0.1% bovine serum albumin), then rinsed twice with ice-cold internalization medium before fixation in paraformaldehyde. For Tf chase experiments, cells were incubated with fluorescently labeled Tf as above, rinsed twice with ice-cold internalization medium, and incubated at 37 °C in normal serum containing medium. At the requested time points, cells were fixed in paraformaldehyde and mounted before image acquisition. For the biochemical analysis of 125I-Tf recycling, confluent transfected cells were cultured for 1 h in the absence of fetal calf serum. Cells were then loaded with iron (Fe3+)-saturated human 125I-Tf (200 ng/ml) for 1 h at 37 °C, washed, and then incubated in fresh medium for the indicated times as previously described (41Mohrmann K. Gerez L. Oorschot V. Klumperman J. van der Sluijs P. J. Biol. Chem. 2002; 277: 32029-32035Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Media were harvested at each time point to assess recycling. Intracellular radioactivity was also determined. 125I-Tf in media and cells was established in a γ-counter. The rate of Rab11 mutants expression was assessed in parallel by fluorescence microscopy and experiments were performed when the expression efficiency reached more than 60% of the cell population. The Structures of Inactive and Active Rab11 Depart from Those of Other Rab Proteins—The GDP/GTP structural cycle of human Rab11a is reported here, including inactive Rab11-GDP at 2Å resolution and active Rab11-GTPγS-Mg2+ at 2.3Å resolution (Table I, Fig. 1). In both crystals forms, Rab11 has the overall fold common to other small GTP-binding proteins including Rab proteins, with a conserved protein core and nucleotide-sensitive switch 1 and switch 2 regions (Fig. 1). The switch regions have well defined conformations in both the inactive and active forms, and undergo large conformational changes that span residues Glu39 to Val46 (switch 1) and Ala68 to Ala79 (switch 2). Other conformational changes are located at the interswitch β-turn (residues Ile53 to Ile60) and reflect its flexibility. The unusually ordered conformation of the switch regions in Rab11-GDP is stabilized in a large symmetrical dimeric interface (Fig. 1A). The buried surface area is ∼2000 Å2, which is larger than average nonspecific interactions between crystal neighbors (42Janin J. Rodier F. Proteins. 1995; 23: 580-587Crossref PubMed Scopus (237) Google Scholar) but in the range (1600 ± 400 Å2) of biologically relevant protein-protein complexes (43Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1764) Google Scholar). 75% of the buried area is contributed by switch 1 and 2 regions, which are therefore essentially inaccessible to protein-protein interactions in the dimer. Rab11-GDP lacks a bound Mg2+ ion at the nucleotide binding site, despite the high Mg2+ concentration in the crystallization conditions (Fig. 1B). The deficiency of stabilizing interactions resulting from the absence of the Mg2+ ion is made up by alternative interactions that maintain the nucleotide tightly bound, including hydrogen bonds of switch 1 with the α-phosphate and the ribose of GDP, and an alternative interaction of the invariant Mg2+-binding Ser from the P-loop (Ser25) with its α-phosphate (Fig. 1B). Furthermore, the invariant switch 1 Thr (Thr43 in Rab11a) that binds Mg2+ and the γ-phosphate of GTP in active Rab11, exchanges a symmetrical hydrogen bond with Glu47 in strand β2 from the symmetrical Rab11 molecule (Fig. 1A). This interaction is strikingly reminiscent of the interaction of the equivalent Thr in small GTP-binding proteins of the Rho family with an aspartate from RhoGDI, which has been proposed to account for the inhibition of guanine nucleotide dissociation by RhoGDI (44Hoffman G.R. Nassar N. Cerione R.A. Cell. 2000; 100: 345-356Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). In contrast, the GTPγS-bound form of Rab11 is monomeric in the crystal. The interactions of the guanine triphosphate nucleotide are similar to those found in other structures of Rab active forms, except that neither Ser20 from the P-loop nor Ser42 from switch 1 interact with the most external γ-phosphate oxygen, which remains accessible to the solvent (Fig. 1C). Switch 2 of Rab11 is the region that diverges most from other GTP-bound Rabs, as it is not helical and it makes less interactions with switch 1 (Fig. 1D). Although an influence of crystal packing cannot be excluded, this unique conformation confirms the trend of Rab proteins in having structurally heterogeneous switch 2 regions in their active forms. The situation is more subtle at the switch 1 region, whose conformation in Rab11-GTP is closest to that in Rab3 and Sec4 despite their sequence divergence (Fig. 1D). Comparison of all structures of GTP-bound Rab proteins shows that switch 1 regions fall in two structural classes, which depend on the main chain conformation of a residue located at the beginning of switch 1 (Ser40 in Rab11) (Fig. 2). This residue either points toward the nucleotide as in Rab11 (Fig. 2A) or is flipped by 180° (Fig. 2B), possibly as the result of a steric conflict with the facing residue from the P-loop (Ser20 in Rab11) for binding the γ-phosphate (Fig. 2, B and C). The alternative conformations of switch 1 determine the shape of a three-dimensional site spanning residues 26–49 in Rab11, which combines switch 1 with an upstream subfamily specific region (α1 and α1-switch 1 loop) and a downstream Rab family motif (switch 1-β2 loop) (Fig. 2). This combination of nucleotide-dependent and Rab subfamily-specific characteristics suggested that this region may define a novel three-dimensional epitope for the interaction of Rab11 with specific partners. To test this hypothesis, Rab11 family-specific residues at the surface of this epitope were changed into their counterparts in Rab3a and tested for their effects on the distribution of early endosomal markers and transferrin transport. The S29F Mutation Unbalances the Distribution of Early Endosomal Markers in HeLa Cells—Residues Ser29 in α1, Arg33 in the α1/switch 1 loop, and Ala49 in the switch 1/β2 loop (Fig. 2A) were mutated to Phe, Asp, and Lys, respectively. The mutants were fused to GFP to examine their cellular localization compared with wild type Rab11 and its classical mutants, and to markers of the endocytic pathway. All mutants strongly labeled membrane structures (Fig. 3, B, C, and F), as also observed for wild type Rab11 (Fig. 3D) or the Q70L mutant (Fig. 3E), but contrasting with the predominantly cytosolic staining of Rab11(S25N) (Fig. 3" @default.
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• W2010236991 date "2004-03-01" @default.
• W2010236991 modified "2023-10-09" @default.
• W2010236991 title "The Structural GDP/GTP Cycle of Rab11 Reveals a Novel Interface Involved in the Dynamics of Recycling Endosomes" @default.
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• W2010236991 doi "https://doi.org/10.1074/jbc.m310558200" @default.
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