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• W2078321316 abstract "The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C2H2 Zn2+ finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C2H2 Zn2+ finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C2H2 Zn2+ finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C2H2 Zn2+ finger play a critical role in the interaction with Rab5. Although the homologous C2H2 Zn2+ finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C2H2 Zn2+ finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C2H2 Zn2+ finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C2H2 Zn2+ finger of EEA1. The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C2H2 Zn2+ finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C2H2 Zn2+ finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C2H2 Zn2+ finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C2H2 Zn2+ finger play a critical role in the interaction with Rab5. Although the homologous C2H2 Zn2+ finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C2H2 Zn2+ finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C2H2 Zn2+ finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C2H2 Zn2+ finger of EEA1. phosphatidylinositol 3-phosphate early endosome antigen 1 surface plasmon resonance glutathione S-transferase N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine As master regulators of membrane trafficking, Rab GTPases cycle between active (GTP-bound) and inactive (GDP-bound) conformations (1Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Google Scholar, 2Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Google Scholar, 3Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Google Scholar). In the active conformation, Rab GTPases interact with diverse effectors implicated in vesicle budding, cargo sorting, motor-dependent transport, tethering, docking, and fusion. Guanine nucleotide exchange factors, GTPase-activating proteins, and other accessory factors, including Rab GDP dissociation inhibitor, provide multiple points of regulation throughout the GTPase cycle by modulating nucleotide binding, GTP hydrolysis, and membrane association (4Segev N. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/OC_sigtrans; 2001/RE11)Google Scholar). Protein kinases and phosphatases have also been implicated in the regulation of Rab function, either directly or by phosphorylation of effectors and regulatory factors (5Ayad N. Hull M. Mellman I. EMBO J. 1997; 16: 4497-4507Google Scholar, 6Bailly E. McCaffrey M. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Google Scholar, 7Prekeris R. Klumperman J. Scheller R.H. Mol. Cell. 2000; 6: 1437-1448Google Scholar, 8Shisheva A. Chinni S.R. DeMarco C. Biochemistry. 1999; 38: 11711-11721Google Scholar). Thus, through regulated interactions with effectors, Rab GTPases couple signal transduction networks to the membrane trafficking machinery. Both fluid phase and receptor-mediated endocytosis depend on activation of Rab5, which plays a critical role in clathrin-coated vesicle formation, endosome motility, and early endosome fusion (9Somsel Rodman J. Wandinger-Ness A. J. Cell Sci. 2000; 113: 183-192Google Scholar). Activated Rab5 interacts with diverse effectors, including scaffolding proteins and tethering factors, and further influences signaling and trafficking events by recruitment of class I and III phosphoinositide 3-kinases to endosomes (10Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell Biol. 1999; 1: 249-252Google Scholar, 11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 12Stenmark H. Vitale G. Ullrich O. Zerial M. Cell. 1995; 83: 423-432Google Scholar, 13Horiuchi 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: 1149-1159Google Scholar, 14Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Google Scholar). The class III phosphoinositide 3-kinase, hVPS34, selectively generates phosphatidylinositol 3-phosphate, PtdIns(3)P,1 which binds to FYVE (Fab1, YOTB/ZK632.12,Vac1, EEA1) and PX (phagocyte oxidase homology) domains in modular signaling and trafficking proteins (15Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Google Scholar, 16Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Google Scholar, 17Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Google Scholar, 18Cheever M.L. Sato T.K. de Beer T. Kutateladze T.G. Emr S.D. Overduin M. Nat. Cell Biol. 2001; 3: 613-618Google Scholar, 19Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Google Scholar, 20Ellson C.D. Gobert-Gosse S. Anderson K.E. Davidson K. Erdjument-Bromage H. Tempst P. Thuring J.W. Cooper M.A. Lim Z.Y. Holmes A.B. Gaffney P.R. Coadwell J. Chilvers E.R. Hawkins P.T. Stephens L.R. Nat. Cell Biol. 2001; 3: 679-682Google Scholar, 21Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Google Scholar, 22Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell Biol. 2001; 3: 658-666Google Scholar, 23Song X. Xu W. Zhang A. Huang G. Liang X. Virbasius J.V. Czech M.P. Zhou G.W. Biochemistry. 2001; 40: 8940-8944Google Scholar). The Rab5 effector early endosome antigen 1 (EEA1) was identified as a lupus autoantigen that localizes to early endosomes (24Mu F.T. Callaghan J.M. Steele-Mortimer O. Stenmark H. Parton R.G. Campbell P.L. McCluskey J. Yeo J.P. Tock E.P. Toh B.H. J. Biol. Chem. 1995; 270: 13503-13511Google Scholar). EEA1 has a modular architecture with an N-terminal C2H2Zn2+ finger, four consecutive heptad repeats, and a C-terminal region containing a calmodulin-binding (IQ) motif, a Rab5-binding site, and a FYVE domain that binds specifically to PtdIns(3)P (15Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Google Scholar, 16Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Google Scholar, 17Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Google Scholar, 25Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Google Scholar). In cell free reconstitution assays, EEA1 is essential for fusion of early endosomes (11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 26Mills I.G. Jones A.T. Clague M.J. Curr. Biol. 1998; 8: 881-884Google Scholar, 27Christoforidis S. McBride H.M. Burgoyne R.D. Zerial M. Nature. 1999; 397: 621-625Google Scholar, 28Simonsen A. Gaullier J.M. D'Arrigo A. Stenmark H. J. Biol. Chem. 1999; 274: 28857-28860Google Scholar). Endosomal localization requires an intact FYVE domain and is sensitive to inhibitors of phosphoinositide 3-kinase activity as well as mutants of conserved residues in the FYVE domain that disrupt PtdIns(3)P binding (15Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Google Scholar, 25Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Google Scholar, 29Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Google Scholar, 30Gaullier J.M. Ronning E. Gillooly D.J. Stenmark H. J. Biol. Chem. 2000; 275: 24595-24600Google Scholar). Localization also requires a region of ∼40 residues proximal to the FYVE domain but is not influenced by mutations that disrupt the interaction with Rab5 (25Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Google Scholar, 31Lawe D.C. Patki V. Heller-Harrison R. Lambright D. Corvera S. J. Biol. Chem. 2000; 275: 3699-3705Google Scholar, 32Lawe D.C. Chawla A. Merithew E. Dumas J. Carrington W. Fogarty K. Lifshitz L. Tuft R. Lambright D. Corvera S. J. Biol. Chem. 2002; 277: 8611-8617Google Scholar). EEA1 forms a parallel coiled coil homodimer, and we have shown that the C terminus of EEA1 has an organized quaternary structure that supports a multivalent interaction with membranes containing PtdIns(3)P, explaining the requirement for the proximal 40 residues (33Callaghan J. Simonsen A. Gaullier J.M. Toh B.H. Stenmark H. Biochem. J. 1999; 338: 539-543Google Scholar, 34Dumas J.J. Merithew E. Sudharshan E. Rajamani D. Hayes S. Lawe D. Corvera S. Lambright D.G. Mol. Cell. 2001; 8: 947-958Google Scholar). Finally, Rab5 also binds to an independent site at the N terminus of EEA1, which has been shown recently (11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 35Kauppi M. Simonsen A. Bremnes B. Vieira A. Callaghan J. Stenmark H. Olkkonen V.M. J. Cell Sci. 2002; 115: 899-911Google Scholar) to bind Rab22 as well. The C2H2 Zn2+ finger of EEA1 shares significant homology with the C2H2Zn2+ finger in the Rab5 effector Rabenosyn as well as the corresponding C2H2 Zn2+ finger in Vac1p, an effector of the yeast Rab5 homologue Ypt51. Like EEA1, Rabenosyn and Vac1p contain a FYVE domain involved in endosome targeting (14Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Google Scholar, 15Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Google Scholar). Temperature-sensitive mutants implicate Vac1p in intervacuolar trafficking and vacuolar protein sorting (36Weisman L.S. Emr S.D. Wickner W.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1076-1080Google Scholar, 37Weisman L.S. Wickner W. J. Biol. Chem. 1992; 267: 618-623Google Scholar). Immunodepletion of Rabenosyn blocks homotypic early endosome fusion as well as heterotypic fusion of endocytic vesicles with early endosomes, suggesting that Rabenosyn plays a critical role distinct from that of EEA1 (14Nielsen E. Christoforidis S. Uttenweiler-Joseph S. Miaczynska M. Dewitte F. Wilm M. Hoflack B. Zerial M. J. Cell Biol. 2000; 151: 601-612Google Scholar). The GTP-bound forms of Rab4 and Rab5 bind to sites in the central and C-terminal regions of Rabenosyn, respectively (38de Renzis S. Sonnichsen B. Zerial M. Nat. Cell Biol. 2002; 4: 124-133Google Scholar). It is not known whether Rab5 binds directly to the C2H2 Zn2+ finger of EEA1, Rabenosyn, or Vac1p. Two-hybrid data indicate that the integrity of the C2H2 Zn2+ finger is essential for Rab5 binding to the N terminus of EEA1 (11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 39Callaghan J. Nixon S. Bucci C. Toh B.H. Stenmark H. Eur. J. Biochem. 1999; 265: 361-366Google Scholar). The requirement for an intact C2H2 Zn2+ finger may reflect a direct interaction with Rab5 or an indirect structural role. For example, the double Zn2+ finger of Rabphilin3A is essential for interaction with Rab3A; however, in the crystal structure of the Rab3A-Rabphilin3A complex, the double Zn2+ finger does not contact Rab3A but instead supports interactions with flanking regions (40Ostermeier C. Brunger A.T. Cell. 1999; 96: 363-374Google Scholar). To gain further insight into the structural basis underlying the function of EEA1 in the tethering and fusion of early endosomes and endocytic vesicles, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C2H2 Zn2+ finger is sufficient for the N-terminal interaction with Rab5C and support a mode of interaction in which an invariant tryptophan residue, which is partially exposed at the interface between the switch I and II regions of Rab5, lies in or near an interface that involves a cluster of hydrophobic residues in the C2H2 Zn2+ finger. EEA1 and Rab5C constructs were amplified with Vent polymerase (New England Biolabs). EEA1 constructs were sub-cloned into a modified pET15b vector containing an N-terminal His6 tag (MGHHHHHHGS). Rab5C constructs were sub-cloned into pGEX-4T1 (Amersham Biosciences) for expression as an N-terminal GST fusion. Site-specific mutants were generated using the QuickChange Site-directed Mutagenesis kit (Stratagene). All constructs and mutants were verified by sequencing the entire coding region from both 5′ and 3′ directions. BL21(DE3)-RIL cells (Stratagene) were transformed with the pGEX-4T1/Rab5C or modified pET15b/EEA1 plasmids, grown in 2× YT-amp (16 g of Bactotryptone, 10 g of Bactoyeast extract, 5 g of sodium chloride, and 100 mg of ampicillin per liter) at 25 (EEA1-(36–91)) or 37 °C (Rab5C and EEA1 constructs) to an A 600 of 0.6, and induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 3 h. For purification of wild type and mutant proteins, cells were suspended in lysis buffer (50 mm Tris, pH 8.0, 0.1 mNaCl, 0.1% mercaptoethanol, 0.1 mmphenylmethylsulfonyl fluoride, 1 mg/ml lysozyme) and disrupted by sonication. Triton X-100 was added to a final concentration of 0.5%, and the cell lysates were centrifuged at 35,000 × gfor 40 min. For His6 fusion proteins, clarified supernatants were loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen). After washing with 10 column volumes of buffer (50 mm Tris, pH 8.0, 500 mm NaCl, 10 mmimidazole, 0.1% mercaptoethanol), His6 fusion proteins were eluted with a gradient of 10–150 mm imidazole. For GST fusions, the supernatants were loaded onto a glutathione-Sepharose column (Amersham Biosciences) equilibrated with 50 mm Tris, pH 8.0, 0.1 m NaCl, 0.1% 2-mercaptoethanol. After washing with 10 column volumes of the same buffer, GST fusion proteins were eluted with 10 mm reduced glutathione. Subsequent ion exchange chromatography using Source Q or Source S (AmershamBiosciences) followed by gel filtration chromatography over Superdex-75 (Amersham Biosciences) resulted in preparations that were >99% pure as judged by SDS-PAGE. To generate the untagged form of Rab5C constructs, GST fusion proteins at a concentration of 2–4 mg/ml were incubated with 2 μg/ml human α-thrombin (Hematologic Technologies) overnight at 4 °C in 50 mm Tris, pH 8.0, 2 mm CaCl2, and 0.1% mercaptoethanol. Following incubation with glutathione-agarose to remove residual fusion protein, the cleaved Rab5C constructs were further purified by ion exchange and gel filtration chromatography. Typical yields of purified proteins range from 10 to 100 mg/liter of bacterial culture. For Rab GTPases, all buffers are supplemented with 2 mmMgCl2. GST-Rab5-(18–185) was exchanged at 37 °C for 30 min with a 25-fold molar excess of GppNHp in 50 mm Tris, pH 8.5, containing 5 mm EDTA, 100 mm NaCl, and 2 units of agarose-immobilized alkaline phosphatase per mg of protein. The exchange reaction was quenched by addition of 10 mm MgCl2, and excess nucleotide was removed by gel filtration on Superdex-75. His6-EEA1 constructs were incubated in a 1:1 molar ratio with GDP-bound or GppNHp-bound GST-Rab5-(18–185) at a concentration of 20 μm for 30 min at 4 °C in buffer A (50 mmTris, pH 8.5, 100 mm NaCl, 2 mmMgCl2, 0.1 mg/ml bovine serum albumin, and 0.1% Tween 20). 50 μl of equilibrated glutathione-Sepharose beads (AmershamBiosciences) were added to 100 μl of the protein mixture and incubated for 1 h. Following centrifugation, the supernatant was collected and the pellet washed three times with 100 μl of buffer A. After washing, the beads were incubated with buffer A containing 10 mm glutathione for 15 min and the fractions analyzed by SDS-PAGE with Coomassie Blue staining. SPR sensograms were collected with a Biacore X instrument (Amersham Biosciences AB) using a carboxy-methylated (CM5) sensor chip to which a GST antibody was covalently coupled using reagents and protocols supplied by the manufacturer. All proteins were dialyzed into flow buffer (10 mm Tris pH 7.5, 150 mm NaCl, 2 mmMgCl2, 0.005% Tween 20) prior to injection. Tandem flow cells were utilized; one was loaded with the 500 nmGST-Rab5C (sample channel), and the other was loaded with an equivalent molar quantity of GST (reference channel) expressed and purified as described above for the GST-Rab5 constructs. GST and GST-Rab5C were injected at a flow rate of 5 μl/min, and subsequent injections were conducted at a flow rate of 20 μl/min. Conversion to the active conformation was achieved by injecting 50 μl of 3 μmRabex-5 followed immediately by a 10-μl injection of 200 nm GppNHp. Binding and dissociation were monitored following 20-μl injections of increasing concentrations of His6-EEA1. Following curve alignment, the reference sensogram, which reflects bulk refractive index changes and/or reversible nonspecific binding, was subtracted from the sample sensogram. The SPR signal at equilibrium (R eq) was extracted from the fit with a simple 1:1 Langmuir binding model and plotted as a function of His6-EEA1 concentration. Dissociation constants (K d) were obtained from a fit to the hyperbolic binding function R eq =R max (His6EEA1)/(K d + (His6 EEA1)), whereR max corresponds to the SPR signal at saturation and is treated as an adjustable parameter. Mean values and standard deviations (ςn−1) were calculated from 2 to 4 independent measurements. Control experiments verify that the fittedK d values are independent of flow rate (5–50 μl/min) and surface coverage (10-fold range), indicating that the equilibrium data are not limited by mass transfer or rebinding. Rab5C at concentrations of 1 or 20 μm in 10 mm Tris, pH 7.5, 150 mm NaCl, 2 mm MgCl2 was titrated with His6-EEA1-(36–91), His6-EEA1-(36–218), or the W104A mutant of His6-EEA1-(36–218). Samples were excited at 290 nm (1 μm Rab5C) or 300 nm (20 μm Rab5C) with a 2 nm bandpass and emission spectra recorded from 300–400 nm (1 nm bandpass) using an ISS spectrofluorimeter. The magnitude of fluorescence quenching (ΔI) at 340 nm was calculated as ΔI =I − I 0, where I andI 0 are the emission intensities in the presence and absence of EEA1, respectively. Values for the dissociation constant (K d) and the number of binding sites (n) were obtained by a non-linear least squares fit to a simple two-state binding model ΔI = ΔI max(b − {b 2 − 4 [EEA1]t/(n[Rab5C]t)}1/2)/2, where b = 1 + [EEA1]t/(n [Rab5C]t) +K d/(n [Rab5C]t), [EEA1]t and [Rab5C]t are the total concentrations of EEA1 and Rab5C, respectively, and ΔI maxcorresponds to the emission intensity at saturation. Concentrations were determined from the absorbance at 280 nm using calculated extinction coefficients ε280(m−1 cm−1) = number of Trp × 5200 + number of Tyr × 1200 + number of Cys × 120. His6 EEA1 constructs were dialyzed against 50 mm Tris, pH 7.5, 150 mm NaCl and centrifuged to equilibrium in an Optima XLI analytical ultracentrifuge (Beckman Instruments). The absorbance at 230 (A 230) or 280 nm (A 280) was measured as a function of the radial distance (r) from the axis of rotation. The x values of the data were transformed as ςm·(r02 −r 2/2), where r 0 was taken as the last point in each data set and ςm was calculated with SEDINTERP (41Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S. Rowe A. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar) using the monomer molecular mass for each construct. Data were compared with the function A(r) =C 0 +C 1·exp(−n·ςm·(r02− r 2)/2), where C 0 andC 1 are constants, and n represents the order of the oligomeric state. The sequence of the EEA1 C2H2 Zn2+ finger was threaded against a protein structure data base using the three-dimensional PSSM fold recognition server to identify a suitable structure for homology modeling. The NMR structure of a C2H2Zn2+ finger from the yeast transcription factor Adr1 (Protein Data Bank code 1paa) was selected for further homology modeling on the basis of its low scoring E value and the absence of gaps in the alignment. The EEA1 C2H2Zn2+ finger shares 29% identity with that of Adr1, which represents the closest homologue of known structure. Non-conserved residues were substituted with the corresponding residues in EEA1, which were modeled in the most frequently observed rotomer conformation compatible with the structure. The resulting homology model represents a rough, working approximation to the actual structure, with an overall fold consistent with the common topology of C2H2 Zn2+ fingers. The active forms of Rab5A, Rab5B, and Rab22 have been shown to interact directly with EEA1-(1–209) (11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 35Kauppi M. Simonsen A. Bremnes B. Vieira A. Callaghan J. Stenmark H. Olkkonen V.M. J. Cell Sci. 2002; 115: 899-911Google Scholar, 39Callaghan J. Nixon S. Bucci C. Toh B.H. Stenmark H. Eur. J. Biochem. 1999; 265: 361-366Google Scholar). This region of EEA1 encompasses a hydrophilic sequence of ∼35 residues, the C2H2 Zn2+ finger, and two consecutive heptad repeats. As shown in Fig.1 A, His6EEA1-(1–218) co-precipitates with GST-Rab5C loaded with the non-hydrolyzable GTP analogue, GppNHp, but does not co-precipitate with the GDP-bound form or in the presence of the Zn2+-chelating agent TPEN. These results are consistent with two-hybrid experiments in which mutation of a cysteine residue involved in Zn2+ coordination disrupts the interaction with constitutively active Rab5A and Rab5B mutants (11Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Google Scholar, 39Callaghan J. Nixon S. Bucci C. Toh B.H. Stenmark H. Eur. J. Biochem. 1999; 265: 361-366Google Scholar). Although these observations demonstrate a requirement for an intact C2H2 Zn2+ finger, it is not clear whether this reflects a direct interaction between the C2H2 Zn2+ finger and Rab5 or whether the C2H2 Zn2+ finger plays an indirect structural role by supporting interactions with flanking regions as is the case for the double Zn2+ finger of the Rab3A effector Rabphilin3A (40Ostermeier C. Brunger A.T. Cell. 1999; 96: 363-374Google Scholar). As shown in Fig. 1, B–D, the interaction of GST-Rab5C-GppNHp with the N terminus of EEA1 can also be detected and quantitatively analyzed by SPR in a BIAcore instrument using a monoclonal GST antibody coupled to a CM5 sensor chip. When injected at concentrations in the low micromolar range, His6EEA1-(1–218) exhibits reversible binding to the GppNHp-bound form of GST-Rab5-(18–185) as judged by the amplitude of the SPR signal compared with the GST reference channel (Fig. 1 B). The signal in the reference channel rises and decays within the response time of the instrument, scales linearly with the concentration of His6 EEA1-(1–218), and therefore represents either a bulk refractive index change or weak, reversible nonspecific binding indistinguishable from a bulk refractive index change. Under the conditions of these experiments, the association of N-terminal EEA1 constructs with GST-Rab5C approaches equilibrium on the time scale of the injection (Fig. 1 C). The quantity bound at equilibrium (R eq) saturates at low micromolar concentrations of His6 EEA1-(1–218) (Fig. 1 D). The data are well fit by a simple Langmuir binding isotherm, yielding a dissociation constant (K d) of 3.3 μm. In contrast, GST-Rab5-(18–185) loaded with GDP shows no detectable binding to His6 EEA1-(1–218), as expected for a bona fideGTPase-effector interaction. An equivalent affinity (K d = 2.3 μm) is observed for the binding of His6 EEA1-(36–218) to full-length GST-Rab5-GppNHp, which includes the hypervariable N- and C-terminal extensions, indicating that the interaction determinants reside within the GTPase domain. To map the minimal interaction site at the N terminus of EEA1 and determine whether the C2H2 Zn2+finger is sufficient for Rab5 binding, SPR experiments were used to analyze quantitatively the binding of GST-Rab5-(18–185) loaded with GppNHp to a panel of His6 EEA1 truncation constructs (Fig.2 A). Elimination of the first 35 residues, corresponding to the hydrophilic N terminus, has no significant effect on the interaction. Likewise, C-terminal truncations eliminating part or all of the heptad repeats show relatively small differences in affinity, which likely reflect systematic variations in the physical properties of the constructs. Indeed, His6EEA1-(36–91), which lacks the N-terminal hydrophilic region and both heptad repeats, binds in a nucleotide-dependent manner to GST-Rab5-(18–185) with an affinity comparable with that of His6 EEA1-(1–218) (compare Fig. 2, B andC, with Fig. 1, C and D). Consistent with this observation, EEA1-(91–218), which lacks the N-terminal hydrophilic region and C2H2 Zn2+finger, shows no detectable binding to GppNHp-bound GST-Rab5-(18–185) at concentrations up to 150 μm (the highest concentration tested). A shorter construct corresponding to the minimal C2H2 Zn2+ finger defined by homology (EEA1-(36–74)) expressed poorly in bacteria and could not be fully purified. Although not suitable for quantitative analysis by SPR, this construct co-precipitates with GST-Rab5-(18–185) loaded with GppNHp but not GDP and, by this measure, does not differ significantly from EEA1-(36–91) (data not shown). We therefore conclude that the C2H2 Zn2+ finger is both necessary and sufficient for the interaction of Rab5C with the N terminus of EEA1. Full-length EEA1 contains over 1200 residues of heptad repeats and forms a parallel coiled coil homodimer in cells (33Callaghan J. Simonsen A. Gaullier J.M. Toh B.H. Stenmark H. Biochem. J. 1999; 338: 539-543Google Scholar). To establish the oligomeric state of N-terminal EEA1 constructs, EEA1-(1–91), EEA1-(36–126), and EEA1-(36–218) were centrifuged to equilibrium in an analytical ultracentrifuge. Whereas EEA1-(1–91) sediments as a uniform monomer at a relatively high concentration of 20 μm, EEA1-(36–126) and EEA1-(36–218) sediment as uniform dimers at a lower concentration of 1 μm (Fig.3 A). Thus, the heptad repeat proximal to the C2H2 Zn2+ finger provides sufficient driving force for stable homodimerization but does not contribute either directly or indirectly to the affinity for Rab5C. Rab5C contains two tryptophan residues (Trp-74 and Trp-114), whereas the N terminus of EEA1 contains a single tryptophan residue (Trp-104). When titrated with His6 EEA1-(36–91), which lacks tryptophan residues, the intrinsic tryptophan fluorescence of untagged GppNHp-bound Rab5-(18–185) undergoes significant quenching accompanied by a small shift in the emission maximum (Fig. 3 B). Both effects saturate at low micromolar concentrations of His6EEA1-(36–91), indicative of a binding interaction. As shown in Fig.3 C, the change in intrinsic tryptophan fluorescence is well described by a simple hyperbolic binding model, which yields aK d of 1.1 μm, in good agreement with the affinity of His6 EEA1-(36–91) for GppNHp-bound GST-Rab5-(18–185) measured by SPR. Consistent with these observations, the intrinsic tryptophan fluorescence of untagged Rab5-GDP is not perturbed by addition of His6 EEA1-(36–91). To determine whether homodimerization influences the stoichiometry of Rab5C binding to the N terminus of EEA1, titration experiments employing intrinsic tryptophan fluorescence to monitor binding were conducted under conditions where the concentration of the fixed component (GppNHp-bound Rab5-(18–185)) was roughly 7-fold greater than the measured K d. The resulting data were analyzed with a titration binding model (see “Experimental Procedures”) that relates the change in intrinsic tryptophan fluorescence to the binding stoichiometry (n), K d, and the maximum" @default.
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• W2078321316 date "2003-03-01" @default.
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• W2078321316 title "Determinants of Rab5 Interaction with the N Terminus of Early Endosome Antigen 1" @default.
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