GDP-bound and Nucleotide-free Intermediates of theGuanine Nucleotide Exchange in the Rab5�Vps9 System*□S

Received for publication, June 7, 2010, and in revised form, August 17, 2010 Published, JBC Papers in Press, September 10, 2010, DOI 10.1074/jbc.M110.152132

Tamami Uejima‡1,2, Kentaro Ihara‡1,3, Tatsuaki Goh§, Emi Ito§, Mariko Sunada§, Takashi Ueda§, Akihiko Nakano§¶,and Soichi Wakatsuki‡4

From the ‡Structural Biology Research Center, Institute of Material Structure Science, High Energy Accelerator ResearchOrganization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan, the §Laboratory of Developmental Cell Biology, Department ofBiological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan, and the ¶Molecular MembraneBiology Laboratory, RIKEN Advanced Science Institute, Saitama 351-0198, Japan

Many GTPases regulate intracellular transport and signalingin eukaryotes. Guanine nucleotide exchange factors (GEFs) acti-vate GTPases by catalyzing the exchange of their GDP for GTP.Here we present crystallographic and biochemical studies of aGEF reaction with four crystal structures of Arabidopsis thali-anaARA7, a plant homolog of Rab5GTPase, in complexwith itsGEF, VPS9a, in the nucleotide-free and GDP-bound forms, aswell as a complex with aminophosphonic acid-guanylate esterand ARA7�VPS9a(D185N) with GDP. Upon complex formationwith ARA7, VPS9 wedges into the interswitch region of ARA7,inhibiting the coordination ofMg2� and decreasing the stabilityof GDP binding. The aspartate finger of VPS9a recognizes GDP�-phosphate directly and pulls the P-loop lysine of ARA7 awayfrom GDP �-phosphate toward switch II to further destabilizeGDP for its release during the transition from the GDP-boundto nucleotide-free intermediates in the nucleotide exchangereaction.

Small GTPases work as a molecular switch, which is turnedoff by its intrinsic GTPase activity hydrolyzingGTP toGDP. Toturn the switch on, the boundGDP should be removed to intro-duce a GTP. Normally, this exchange reaction is much slowerthan the rate of intrinsic GTPase activity because nucleotidebinds to small GTPase tightly with the Mg2� ion. GEF5enhances the nucleotide exchange of its cognate GTPase bydestabilizing the Mg2� ion and GDP binding to the GTPase.Recognition of the GDP-bound GTPase by GEF is a critical but

transient step prior to the formation of a nucleotide-freeGTPase�GEFbinary complex (1, 2). Because the nucleotide-freecomplex is stable in vitro, most GTPase�GEF complexes havebeen crystallized and structurally analyzed as the nucleotide-free form in the past, with a few exceptions containing nucleo-tides. One of them is a structure of eFF1A�eEF1B� in complexwith different di- and triphosphate nucleotides, inwhich�- and�-phosphate electron densities of the nucleotides were ambig-uous and only GMP could have been modeled (3). Anotherexample is the Arf1�Sec7 complex, in which introducing eitheran abortive inhibitor or amutation on theGEF allowed forGDPbinding into the Arf1�Sec7 complex (4, 5). The complex struc-ture between ROP4�GDP, a member of the plant Rho family,and its GEF PRONE8 indicated that GDP bound to theROP4�PRONE8 complex loosely (6). A complex betweenCdc42and the DHR2 GEF domain of DOCK9 GEF has been crystal-lized in three different nucleotide forms: nucleotide-free, GDP-bound, and GTP/Mg2�-bound. These structures demonstratethat the nucleotide sensor found in the �10 helix of the DHR2domain is responsible for the exclusion of GDP/Mg2� and theintroduction of GTP/Mg2� (7). It should be noted that none ofthe nucleotides were directly recognized by the GEFs in thecomplex structures mentioned above. These exceptions haveencouraged us to investigate the transient nucleotide recogni-tion in the GEF-catalyzed reaction.Rab small GTPases regulate vesicular transport in eukaryotes

(8, 9), including plants (10). ARA7, an Arabidopsis homolog ofmammalian Rab5, controls endosomal fusion (11) and is spe-cifically activated by VPS9a, an Arabidopsis GEF for ARA7,which contains a Vps9 domain (12, 13). To understand thedetailed mechanisms of the initial reaction pathway of the GEFcatalyzed guanine nucleotide exchange, in particular the desta-bilization mechanisms responsible for the release of GDP forwhich no direct structural data are available (8), we have usedprotein crystallography and biochemical assays to identify thesmall GTPase�GEF intermediates in Rab small GTPases. TheARA7�VPS9a complex was crystallized in three different nucle-otide states: the nucleotide-free form, the GDP-bound form,and the GDP analog, GDPNH2-bound form. In addition, weobtained the structure of the ARA7�GDP�VPS9a(D185N)mutant to compare with the GDPNH2-bound form. The fourcomplex structures provide a mechanistic description of theintermediates of guanine nucleotide exchange in Rab5�Vps9, inwhich GEF interacts with the nucleotide directly and GDP is

* This work was supported by a Protein 3000 Project grant and a Grant-in-Aidfor Scientific Research from the Ministry of Education, Culture, Sports, Sci-ence, and Technology of Japan.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables 1 and 2 and Figs. 1–10.

The atomic coordinates and structure factors (codes 2EFC, 2EFD, 2EFE, and 2EFH)have been deposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).

1 Both authors contributed equally to this work.2 To whom correspondence may be addressed. Tel.: 81-29-864-5631; Fax:

81-29-879-6179; E-mail: tamami.uejima@kek.jp.3 To whom correspondence may be addressed. Tel.: 81-29-864-5631; Fax:

81-29-879-6179; E-mail: kentaro.ihara@kek.jp.4 To whom correspondence may be addressed. Tel.: 81-29-864-5631; Fax:

81-29-879-6179; E-mail: soichi.wakatsuki@kek.jp.5 The abbreviations used are: GEF, guanine nucleotide exchange factor;

GDPNH2, aminophosphonic acid-guanylate ester; GppNHp, guanosine-5�-(�,�-imido)triphosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 47, pp. 36689 –36697, November 19, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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released by an interplay of the two conserved residues, theaspartate finger of VPS9a and the P-loop lysine of ARA7.

EXPERIMENTAL PROCEDURES

Proteins—Amino acid sequences of ARA7 and VPS9a wereobtained from the Arabidopsis thaliana genome data base inthe MIPS (Munich Information Center for Protein Sequences,available on the World Wide Web), with accession codesAt4g19640.1 and At3g19770.1, respectively. ARA7 (residues1–179) and VPS9a (residues 1–265) or mutants of VPS9a wereproduced as glutathione S-transferase (GST)-fused proteinsusing the pGEX4T1 expression vector (GEHealthcare) in Esch-erichia coli strains DH5� and Rosetta-gami(DE3)pLysS (Nova-gen), respectively. The GST domain was cleaved by thrombin.ARA7 was purified in the GDP-bound form in the absence ofMg2�, without the addition of GDP. After mixing ARA7�GDPand VPS9a, nucleotide-free ARA7�VPS9a was separated usingan anionic exchanger followed by size exclusion chromatogra-phy in a solution containing 10 mM Tris-HCl, pH 7.4, 50 mM

NaCl, and 1 mM EDTA. Both selenomethionine-labeled ARA7andVPS9a were expressed in the E. coli strain B834(DE3)pLysS(Novagen), mixed, and purified as described above.Pull-down Assay—Mutations in ARA7 and VPS9a were

introduced by PCR-based mutagenesis. Protein extracts fromyeast cells expressingWTormutantARA7under the control ofthe constitutive TDH3 promoter were incubated with GST-VPS9a or GST, which were both prebound to the glutathione-Sepharose 4B resin (GE Healthcare). After incubation, the res-ins were washed three times, separated by SDS-PAGE, andanalyzed by immunoblot using the anti-ARA7 antibody.Yeast Two-hybrid Assay—The cDNAs encoding the wild type,

the constitutively active (Q69L), the dominant negative (S24N),and the nucleotide-free (N123I) forms of ARA7 were subclonedinto pAD-GAL4–2.1 (Stratagene). The ORF of VPS9a was sub-cloned into pBD-GAL4-GWRFC by the LR reaction (Invitrogen).Theconstructswere co-transformed into theAH109 strain (Clon-tech), and interactionswere testedby spotting the samenumberofco-transformed yeast cells on His plates. At least three indepen-dent transformants were tested for each interaction. Empty vec-tors were co-transformed for the negative controls.Crystallography—Crystals of nucleotide-free ARA7�VPS9a

were obtained from a mixture of 12 mg/ml nucleotide-freeARA7�VPS9a and an equal amount of a reservoir solution con-taining 3% PEG 4000, 50 mM imidazole malate, pH 5.5, 50 mM

NaCl, and 30 mM �-mercaptoethanol equilibrated against thereservoir solution by hanging drop vapor diffusion at 16 °C.Molecular replacement, using the structures of Rab5 andRabex-5, failed. Therefore, nucleotide-free selenomethionineARA7�VPS9a was crystallized under the same conditions. As acryoprotectant, 20% ethylene glycol was added to the reservoirsolution. Crystals of ARA7�GDPNH2�VPS9a were obtainedfrom a mixture of 2.5 mg/ml nucleotide-free ARA7�VPS9awith 43 mM guanosine-5�-(�,�-imido)triphosphate (GppNHp)(Sigma) and an equal quantity of the reservoir solution contain-ing 20% PEG 3350, 200 mM lithium citrate, pH 7.5, and 10 mM

DTT, equilibrated against the reservoir solution by hangingdrop vapor diffusion at 16 °C. ARA7�GDP�VPS9a and ARA7�GDP�VPS9a(D185N) were crystallized in the same manner as

the ARA7�GDPNH2�VPS9a complex except that 1 mM GDP, in-stead of 43mMGppNHp, was used. Crystals of nucleotide-boundARA7�VPS9awere frozenwithout adding cryoprotectant.Diffrac-tion data were collected at the Stanford Synchrotron RadiationLightsource, SPring-8, and the Photon Factory. The crystallo-graphic data and refinement statistics are summarized in supple-mental Figs. 11 and 12. The diffraction data were processed usingHKL2000 (14). The initial phase set of the nucleotide-freeARA7�VPS9a crystal data were calculated using SOLVE/RESOLVE(15), followedby iterative automatic andmanualmodelrefinement by REFMAC5 (16) in the CCP4 suite (17) with COOT(18) or O (19), respectively. Molecular replacement by MOLREP(20) in the CCP4 suite gave an initial model of the other crystaldata using the coordinates of the nucleotide-free selenomethi-onine ARA7�VPS9a. Themodel was refined as described above,with the help of wARP (21). Structural figures were prepared byRASMOL (22), MOLSCRIPT (23), and RASTER3D (24). Thestereochemical quality of the protein structures was checked byPROCHECK (25), and no main-chain torsion angels werelocated in the disallowed regions of the Ramachandran plot.The crystallographic data and refinement statistics are summa-rized in supplemental Figs. 11 and 12. The electron densitymaps around the nucleotides and in the surrounding residuesare shown in supplemental Fig. 5.GEF Activity—Single turnover guanine nucleotide exchange

events on ARA7 GDP were initiated by adding 100 �M

GppNHp to a solution containing 0.5 �M ARA7 GDP in 20 mM

Tris-HCl, pH 8.0, 150 mM NaCl, and 0.5 mM MgCl2 at 25 °C,with 0.0–1.5 �M VPS9a and its three mutants, D185A, D185N,and Y225A. Nucleotide exchange of Ara7 from GDP toGppNHpwas detected as a decrease in the intrinsic tryptophanfluorescence of ARA7, reflecting the state of its nucleotides,following the procedures used for Rab5 and Rab7 (26). Trypto-phan fluorescence was excited at 290 nm and detected at 340nm in a fluorescence spectrophotometer (F-2500, Hitachi).Data were collected for 700 s following the addition ofGppNHp. The pseudo-first order rate constants, kobs, wereobtained by fitting the decrease in intrinsic tryptophan fluores-cence to a sum of single exponentials. Each kobs was measuredthree times, whereas the intrinsic nucleotide exchange rate ofARA7 without VPS9a, kint, was measured only once and wasdetermined to be 3.9 � 10�3 s�1. The kobs value for the wild-type VPS9a was fit to a pseudo-Michaelis-Menten hyperbolicfunction to determine the apparent kcat and Km, whereas thevalues of kobs for the VPS9a mutants were obtained from theslopes corresponding to the GEF efficiencies, kcat/Km.Surface Plasmon Resonance—For determination of binding

affinities between GST-fused VPS9a and ARA7, plasmon reso-nance experiments were carried out using a BIAcoreTM 2000system (BIAcore) using a sandwich assay with anti-GST anti-body to immobilizeGSTorGST-fused proteins onto the sensorchip CM5 (BIAcore). All data collection was performed in theHBS-EP buffer, containing 10 mM HEPES (pH 7.4), 150 mM

NaCl, 3mMEDTA, and 0.005% surfactant P20 (pH 7.4) (BIAcore),and the HBS�Mg2�P buffer, containing 10 mM HEPES(pH7.4), 150mMNaCl, 1mMMgCl2, and 0.005% surfactant P20(pH 7.4) (BIAcore). Values of the dissociation constants (KD)were computed with BIAcoreTM software (BIAcore), and the

Crystal Structures of ARA7�VPS9

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final KD values were calculated as the average of threemeasurements.Preparation ofGDPNH2—GppNHpwas dissolved inwater to

a concentration of 400 mM and then boiled for 10 min at 95 °C.The purity of GDPNH2 and GppNHp was checked by anionicexchange chromatography.

RESULTS

Interaction between VPS9a and ARA7—The four crystalstructures showed remarkable similarities in the overall config-urations of VPS9a and ARA7, suggesting that the complexremains largely intact during GDP release from ARA7 (Fig. 1and supplemental Fig. 1, a–c). Small GTPases have two regions,switches I and II, which are structurally different in the GTP

and GDP-bound forms (27). VPS9auses two �-helices, �V4 and �V6,and three loops, �V1/�V2, �V3/�V4, and �V6/�C, to recognize theP-loop, interswitch region, andswitch II of ARA7 (supplementalFig. 2, a–d), in contrast to the otherGEFs for Rab, MSS4 (28), and Sec2(29), which recognize their cog-nate Rab GTPases mainly throughthe switch I region. Interactionsseen in the ARA7�VPS9a crystalswere confirmed by the GST pull-down experiments (Fig. 2). ARA7mutants, V36P, T42A, and G44P,whose residues belong to switch I,interact with VPS9a as tightly as theGDP dominant mutant S24N,which was known to interact withVPS9a strongly both in vivo and invitro. On the other hand, severalother ARA7 mutants whose resi-dues are conserved in the switch IIand interswitch regions, A46D,F47D, W64A, A67G, Q69E, S74A,L75A, M78A, and Y79A, could notbind to VPS9a, which suggestedthat these residues in switch II andthe interswitch region were criticalin binding to VPS9a, and thismutational analysis of ARA7 wasconsistent with the observation ofthe specific interactions in theARA7�VPS9a crystal structure. Inparticular, there were two sets ofcritical interactions holding ARA7and VPS9a together; one was ahydrophobic interaction of VPS9aTyr225 with the interswitch regionof ARA7, and the other was an elec-trostatic interaction between thetwo conserved residues, VPS9aAsp185 and ARA7 Lys23 (supple-mental Fig. 12, a–d). VPS9a Tyr225

makes three van der Waals contacts with ARA7 Ala46, Phe47,and Trp64 at the bottom of the interswitch region of ARA7(Fig. 3). By comparing the structures of ARA7�GDP�VPS9a andRab5A�GppNHp�Mg2� (30) or the form B of Rab5A�GDP�Co2�

(31), we found that the hydrophobic contacts partiallyunzipped the antiparallel �-sheet (�2/�3) of ARA7 in the inter-switch region to widen the nucleotide binding pocket, asobserved in Arf1�Gea2 (32) (Fig. 4, a and b). Structural changesof ARA7 upon binding to VPS9a were largely confined to theswitch regions: switch I in ARA7�GDP�VPS9a opened, andswitch II partially rearranged. As a result, GDP also shiftedalong its long axis, by 0.5 Å, away from the interaction regionwith VPS9a (Fig. 4, a and b). Another striking effect of the bind-ing of VPS9a to ARA7 was to removeMg2� from ARA7, which

FIGURE 1. Structure of ARA7�GDP�VPS9a. ARA7�GDP�VPS9a is colored based on the secondary structureslabeled in supplemental Fig. 3, a and b. GDP is drawn as a stick model with small spheres to indicate carbon(gray), oxygen (red), and phosphorus (magenta) atoms.

FIGURE 2. Interactions between VPS9a and mutants of ARA7. Some mutations in the ARA7 structureseverely affected the interactions with VPS9a, as predicted from the three-dimensional structure. The quantityof the ARA7 protein contained in 10% of the yeast lysate used in each assay is shown as an ARA7 input.

Crystal Structures of ARA7�VPS9

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would lead to the destabilization of GDP binding in theARA7�VPS9a complex. Indeed, none of the ARA7�VPS9a com-plex structures containedMg2�. Moreover, Mg2� could not beintroduced into any of the crystals reported here by co-crystal-lization. IntroducingMg2� ion in the nucleotide-free andGDP-bound ARA7�VPS9a structures at the position corresponding

to that of Rab5A�GDP structure (the form B of 1TU4) (31)would produce severe steric hindrance between one of thecoordinating water molecules, W4, and one of the oxygenatoms of VPS9a Asp185 (Fig. 4).Comparison with Rabex-5�Rab21 Complex Structure—The

structures of VPS9a, in each of the four complexes presented inthis study, resembled that of the apo-form human Rabex-5Vps9 domain described previously (33). The root mean squaredeviation between 184 corresponding C� positions of VPS9aand Rabex-5 was 1.5 Å, despite the low sequence identity, 27%(supplemental Fig. 3a), between the two domains (Fig. 5).Superposition of the ARA7�VPS9a structure with the apo-formRabex-5 structure (Fig. 5) revealed an intriguing possibility ofside-chain movements of two key residues of VPS9a, Asp185

and Tyr225, corresponding to Asp313 and Tyr354 of Rabex-5.These movements introduce additional interactions withARA7: three polar interactions with VPS9a Asp185 and threevan der Waals contacts with VPS9a Tyr225 (Fig. 5 and supple-mental Fig. 2, a–d). The N-terminal helical structure was con-served between the two molecules, despite the low sequenceidentity of the N-terminal regions, 19% for the first 97 residues(supplemental Fig. 3a), and the lack of �HB1 in VPS9a in thestructure.Aspartate Finger of VPS9a and P-loop Lysine of ARA7—The

most unique and direct ARA7 recognition was provided byVPS9a Asp185 near the �-phosphate of GDP and ARA7 Lys23.This corresponds to the invariant aspartate residue in Vps9-containing proteins, which has been described as crucial forGDP release fromRab5 and, accordingly, termed the “aspartate

FIGURE 3. Hydrophobic contacts between ARA7 and VPS9a Tyr225. Theside chain of Tyr225 on the �V6 of VPS9a (cyan) makes van der Waals contacts(yellow dots) with the side chains of Ala46, Phe47, and Trp64 in the interswitchregion of ARA7 (magenta). The side chain of Asp185 on the �V4 of VPS9a is alsoshown.

FIGURE 4. Expected structural changes of ARA7 upon binding to VPS9a. a and b, the form B of Rab5A�GppNHp�Mg2� (Protein Data Bank entry 1R2Q; green)and Rab5A�GDP�Co2� (chain B of Protein Data Bank entry 1TU4; green), respectively, are superimposed on the structure of ARA7�GDP (magenta) bound to VPS9a(cyan). A subset of the VPS9a structure, including Asp185 and Tyr225, whose side chains are drawn as ball-and-stick models, is displayed for clarity. Nucleotidesin Rab5A and ARA7 are drawn as ball-and-stick models and colored in light green and pink, respectively. Structural differences between the switches and thephosphate-binding cassette are indicated by green-to-magenta arrows. The interswitch region of ARA7 is unzipped by hydrophobic contacts with VPS9a (Fig.3), and the unzipping is represented by the C� positions of the ARA7 Ala45 and Rab5A Ala55, indicated by orange spheres and an arrow. The side-chain oxygensof VPS9a and the oxygens coordinated to Co2� (blue-gray sphere), or Mg2� (gray sphere), of Rab5A are drawn as red spheres with red dots indicating coordinationto the metal.

Crystal Structures of ARA7�VPS9

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finger” (33), in analogy with the “glutamate finger” correspond-ing to the invariant glutamate in Sec7 proteins, such as Gea2(32) or ARNO (34). On the other hand, ARA7 Lys23 is con-served in many GTPases as part of the diphosphate bindingloop containing four consensus residues in an eight-residuesequence, GXXXXGK(T/S) (22). This conserved lysine residuecontacts and neutralizes the �-phosphate of GDP in the com-plex (supplemental Fig. 4a). A side-chain oxygen of the VPS9aAsp185 was stabilized by a hydrogen bond to the main-chainNH of ARA7 Gly68 in switch II of ARA7. The carboxyl oxygen

atoms of VPS9a Asp185 also formed electrostatic interactionswith the side-chainNH3

� of ARA7 Lys23 (supplemental Fig. 4a).Interestingly, a side-chain oxygen of the glutamate finger, Gea2Glu654, also forms a salt bridge with Arf1 Lys30, correspondingto ARA7 Lys23.Crystal Structures of Two Complexes, ARA7�GDPNH2�VPS9a

and ARA7�GDP�VPS9a(D185N)—Destabilization of GDP wasstructurally investigated using two crystal structure variants,ARA7�GDPNH2�VPS9a and ARA7�GDP�VPS9a(D185N). Thecrystal structure of ARA7�GDPNH2�VPS9a was accidentallyobtained during crystallization trials of ARA7 in the GTP-bound form, in which an excess of GppNHp was added tothe nucleotide-free ARA7�VPS9a solution. The structure ofARA7�GDPNH2�VPS9a did not exhibit significant overallstructural changes compared with that of the nucleotide-freeARA7�VPS9a (supplemental Fig. 1, a and b). The electron den-sity corresponding to the GDPNH2 molecule, a product ofGppNHp hydrolysis, was clearly observable and excluded thepossibility of residual GppNHp (supplemental Figs. 5 and 6b).The shape of the electron density itself did not provide evidencefor the preferential binding of GDPNH2 over GDP at the cur-rent resolution of the complex structures, because the hydro-lysis of GppNHp potentially produces either GDP or GDPNH2.However, an anion exchange chromatography analysis with afresh GppNHp solution indicated the presence of GDPNH2,most likely as a hydrolysis product. Moreover, we established aprotocol for the almost complete conversion of GppNHp toGDPNH2 by simply boiling the former in the absence of Mg2�

(supplemental Fig. 7, a and b). The addition of 5 mM GDPNH2to the nucleotide-free ARA7�VPS9a complex solution resultedin reproducible crystal growth. In contrast, an addition of 5mM

GDP to the same solution caused precipitation of the proteins.These results suggested that our ARA7�GDPNH2�VPS9a had

nothing but GDPNH2.Compared with the complex

structure ofARA�GDP�VPS9a, therewas an interesting difference. Thedistance between the side chain ofAsp185 in VPS9a and the �-phos-phate of GDP in the ARA7�GDPNH2�VPS9a is shorter thanthat in the ARA�GDP�VPS9a (Fig. 6,a and b). This observation hasprompted us to investigate the pos-sibility of an inhibitory effect in theGEF reaction by GDPNH2. How-ever, the GEF activity assay showedno effective inhibition of GEF activ-ity in vitro for the addition of up to50 �M GDPNH2 (data not shown).To further analyze the effect of inhi-bition of nucleotide exchange, theD185N mutant of VPS9a wasdesigned based on the GDPNH2-boundARA7�VPS9a complex struc-ture. We anticipated that the inter-action mode and affinity betweenVPS9a(D185N) and ARA7�GDP

FIGURE 5. Superposition of human Rabex-5 to VPS9a. Apo-form of humanRabex-5 (Protein Data Bank entry 1TXU; orange) is superposed toARA7�GDP�VPS9a (magenta and cyan). The sequence identity between Vps9of Rabex-5 and VPS9a is low, 27% (supplemental Fig. 3a). The root meansquare deviation between 184 corresponding C� positions of VPS9a andRabex-5 was 1.5 Å. Possible structural shifts upon the complex formation areindicated by orange-to-cyan arrows.

FIGURE 6. Polar interactions around ARA7 Lys23. a– d, oxygens (red) or a nitrogen (blue), surrounding theside-chain NH3

� of ARA7 Lys23 in ARA7�GDP�VPS9a, ARA7�GDPNH2�VPS9a, nucleotide-free ARA7�VPS9a, andARA7�GDP�VPS9a(D185N), respectively, are shown. The side-chain NH3

� of ARA7 Lys23 is surrounded by a �-po-sition oxygen of GDP, side-chain COO� (ARA7 Asp65 and VPS9a Asp185), and main-chain carbonyl oxygens(Gly17, Asp18, and Thr66 of ARA7). Possible hydrogen bonds and electrostatic interactions are shown with cyandots and magenta dots, respectively. Distances exceeding 3.5 Å are shown as black dots. InARA7�GDP�VPS9a(D185N), a water molecule is located between the side-chain NH3

� of ARA7 Lys23 and the�-position oxygen of GDP. A possible hydrogen bond between the side-chain COO� of VPS9a Asp185 andthe �-position oxygen of GDP is highlighted by green dots.

Crystal Structures of ARA7�VPS9

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would be almost the same as those of ARA7�GDPNH2�VPS9aor would be stronger than those of ARA7�GDPNH2�VPS9a,except that the polarity of the hydrogen bond between the�-phosphate and the VPS9a(D185N) Asn185 would be oppo-site compared with the case between GDPNH2 and VPS9aAsp185 in ARA7�GDPNH2�VPS9a. Whereas the crystal struc-ture of the ARA7�GDP�VPS9a(D185N) complex confirmedmost of these, it also reveals the existence of a new water mol-ecule betweenGDP�-oxygen andNH3

� of ARA7 Lys23 possiblydue to the repulsion between the side-chain NH2 of VPS9aAsn185 and NH3

� of ARA7 Lys23 (Fig. 6d). Interestingly, thebinding of GDP in the ARA7�GDP�VPS9a(D185N) mutant isweaker than in the wild-type ARA7�GDP�VPS9a, as judgedfrom their higher temperature factors (supplemental Fig. 8, aand d).Following these crystallographic observations, we then

investigated GEF activities and binding affinities of theVPS9a(D185N) mutant. Surprisingly, this mutant exhibited ahigher GEF activity in the presence of Mg2� (Fig. 7). We there-fore prepared additionalmutants, D185A and Y225A, of VPS9aandmeasured their GEF activities. As expected, the D185A andY225A mutants exhibited much lower GEF activities, reducedby 12 and 25 times, respectively, compared with the wild-typeVPS9a. In contrast, the GEF efficiency of VPS9a(D185N) wasonly 1.3 times lower than that of the wild-type VPS9a with anapparently higher maximum rate constant (Fig. 7).

The mutational analysis was further extended with addi-tional VPS9a mutants, A184K, D185E, and D185A/Y225A. Apull-down assay of these mutants showed that ARA7 could notinteract with any other VPS9a mutants except for theVPS9a(D185N). Only this mutant showed rather weak bindingto ARA7 (Fig. 8a). Furthermore, a surface plasmon resonanceexperiment was performed to measure the dissociation con-stants (KD) between VPS9amutants and ARA7 using BIAcoreTM2000 (BIAcore). The D185N mutant of VPS9a was shown tohavemuch higher dissociation constants (KD) than that of wild-type VPS9a, increased by factors of 4.9 and 3 with Mg2� andEDTA, respectively (Fig. 8b), although these KD values of theD185N mutant were smaller than those of other VPS9amutants, D185A and Y225A. This result is consistent with thepull-down assay. These results corroborated well with thenotion that theD185Nmutant ofVPS9a had a highGEF activitydespite the weaker binding for ARA7 and that this high GEFactivity of D185N mutant might have come from instabilityof GDP, which is observed in the crystal structure ofARA7�GDP�VPS9a(D185N).

DISCUSSION

Complex Formation—In the pull-down and yeast two-hybridassays, VPS9a interacted with either the dominant negative(S24N) or the nucleotide-free (N123I) ARA7 but not withthe constitutively active GTP-bound form (Q69L) (Fig. 9). TheN-terminal catalytic domain (residues 1–265) showed essen-tially the same results as the full-length VPS9a (data notshown). Interestingly, there was a significant differencebetween the results of the yeast two-hybrid and pull-downexperiments. VPS9a interacted with the wild-type ARA7 in thepull-down experiment but not in the yeast cells. Anionexchange columnchromatography demonstrated that the puri-fied wild-type ARA7 used in the pull-down experiment wascompletely in theGDP form (data not shown), which suggestedthat GTP in the wild-type ARA7 had been hydrolyzed to GDPby the intrinsic GTPase activity during purification. On theother hand, the wild-type ARA7 in the yeast two-hybrid exper-iment was expected to be present predominantly in the GTP

FIGURE 7. GEF activity of VPS9a. The pseudo-first order rate constants areplotted against the concentrations of the wild-type and three mutants ofVPS9a. For the wild-type VPS9a, the maximum rate constant and the apparentdissociation constants are estimated. Values of the rate constants are to becompared with the intrinsic nucleotide exchange rate of ARA7 in the absenceof VPS9a, kint, 3.9 � 10�3 (1/s). Values of the dissociation constants are sum-marized in the table below the plots.

FIGURE 8. Interactions between ARA7 and mutants of VPS9a. a, mutationsin the conserved Asp185 and Tyr225 of VPS9a strongly affect the interactionbetween ARA7 and VPS9a. Note that the D185N mutation of VPS9a does notcompletely abolish the interaction, which is consistent with the results shownin Fig. 4. The quantity of ARA7 protein contained in 10% of the yeast lysateused in each assay is shown as an ARA7 input. b, dissociation constants (KD)between ARA7 and VPS9a with 1 mM Mg2� or 1 mM EDTA buffer.

Crystal Structures of ARA7�VPS9

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form because the concentration of GTP is higher than that ofGDP in the cell cytosol. Similar behaviors have been observed inother plant orthologs of Rab5, RHA1, and ARA6 (13). Thus, weconclude that the difference between the pull-down and yeasttwo-hybrid experiments for the interaction of VPS9a with thewild-type ARA7 reflects the two distinct nucleotide states.Protonation of GDP �-Phosphate—We present here four

complex structures of plant Rab5 and its GEF, which indicateseveral unexpected features. The first is the observation of anunusually close contact, 2.9–3.1 Å, between a �-phosphateoxygen of GDP and a side-chain oxygen of VPS9a Asp185 (Fig.6a). This distance is too short for two negatively charged oxy-gen atoms and suggests that it is likely that one of these twooxygen atoms is protonated to form a hydrogen bond. Pro-tonation of nucleotides can be modulated by the surround-ing electronic environment. The pKa for GDP is 6.7 (35), andGDP alone in solution is largely deprotonated at pH 7.5, theconditions under which the ARA7�GDP�VPS9a complexeswere crystallized. However, the pKa for GDP in ARA7�VPS9ashould be higher due to the interaction with the side-chainCOO� of VPS9a Asp185. An example of such a pKa increasehas been reported, for example, for the G12D mutant of Raswith an increase of 1.4 pH units in the �-phosphate pKa inRas(G12D)�GppNHp�Mg2� relative to Ras(G12)�GppNHp�Mg2�,resulting in the protonation of GppNHp (36). Such a pKa increasecaused by the negative charges is also consistent with the pKaincrease of the nucleotides caused by the removal of Mg2�, assuggested by quantum mechanical calculations (35, 36), or withthe arginine finger ofRasGAP, predicted by computer simulations(37). These observations support the notion that a similar pKaincrease of GDP would be caused by the presence of VPS9aAsp185 and the absence ofMg2� in the vicinity and result in theprotonation of the GDP �-phosphate in ARA7�GDP�VPS9a,leading to the short distance between the �-phosphate oxygenof GDP and the side-chain oxygen of VPS9a Asp185.

GDP Analog GDPNH2 and VPS9a(D185N) Mutation—Sec-ond, a comparison of the two ternary complex structures,ARA7�GDPNH2�VPS9a andARA7�GDP�VPS9a(D185N) gives afurther insight into the stability of GDP in the complexes. Wehave shown that the distance between the �-phosphate andVPS9a Asp185 in ARA7�GDPNH2�VPS9a was shorter than thatin ARA7�GDP�VPS9a (Fig. 6, a and b) and that VPS9a(D185N)has higher GEF activity due to the instability of GDP (Fig. 6d).We at first anticipated that VPS9a(D185N) mutation wouldhave the same effect as the GDP analog, GDPNH2, which canprovide the NH2 base to the �-phosphate oxygen and block thenucleotide exchange completely with stronger affinity between�-phosphate of GDP and the substituted Asn185 residue ofVPS9a. However, the structure of the D185Nmutant of VPS9aturned out to be counter to our expectations; as mentionedabove, the introduction of the new water molecule betweenGDP �-oxygen and NH3

� of ARA7 Lys23 destabilized GDP, andaccordingly, thisD185Nmutant obtained a higherGEF activity.Based on these observations, design and search for new chem-ical compounds mimicking the nucleotide analogues, whichwould block the GEF reaction efficiently, are being pursued.Initial Pathway of the GEF Reaction—Third, these four com-

plex structures shed light on the initial GEF exchange reactionpathway. The Rab5 GEF destabilizes GDP by modifying thelocal environment of the �-phosphate with one of the carboxyloxygen atoms of the aspartate finger, VPS9a Asp185. This pro-cess is coupled with the movement of the side chain of ARA7Lys23. The electrostatic interaction between GDP �-oxygenand NH3

� of ARA7 Lys23 is observed in the ARA7�GDP�VPS9astructure (Fig. 6a). On the other hand, it is absent in the othertwo complex structures; the distance is much longer in theARA7�GDPNH2�VPS9a structure, and the water molecule isinserted in the ARA7�GDP�VPS9a(D185N) structure (Fig. 6, band d). Taken together, VPS9a appears to use three steps toincrease the pKa of the �-phosphate in GDP-bound ARA7,leading to the putative protonation of the �-phosphate of GDP:1) removing Mg2� from GDP, 2) placing the side-chain COO�

of VPS9a aspartate finger in the vicinity of the �-phosphate ofGDP, and 3) destabilizing the electrostatic interaction betweenthe side-chain NH3

� of ARA7 Lys23 and GDP by moving theP-loop lysine fromGDP toward switch II Glu65. As a net result,deprotonation of the GDP �-phosphate oxygen is induced,whichwould destabilize theGDP and enhance its release due torepulsion between the two oxygen anions of GDP and VPS9aAsp185 (supplemental Fig. 9, a–f). In the following, we will dis-cuss these three steps with reference to the other smallGTPase�GEF complex structures.Removal of Mg2� Ion—As one of the first steps of the GEF

reaction, theMg2� ion is removed from the nucleotide bindingsite of small GTPase�GEF complexes. The exclusion of theMg2� ion leads to a decrease in the nucleotide affinity of thenucleotide binding site. This is accomplished in three distinctways by various conserved residues: switch II alanine of Ras inRas�SOS (38) and Rac in Rac�Tiam1 (39), valine of nucleotidesensor in DOCK9 (7), glutamate finger of Sec7 (32, 34), andaspartate finger of VPS9a (this work)(33). Interestingly, Ala27 ofArf�Gea2 (32), which corresponds to the switch II alanine in Rasand Rac, is flipped outward, and instead the glutamate finger of

FIGURE 9. Nucleotide-dependent interactions between ARA7 and VPS9a.Pull-down and yeast two-hybrid assays indicated that VPS9a preferred thedominant negative (S24N) or the nucleotide-free (N123I) forms of ARA7 tothe constitutively active (Q69L) form. The quantity of the ARA7 proteincontained in 10% of the yeast lysate used in each pull-down assay isshown (ARA7 input). AD and BD were fused to the transcription activatingdomain and DNA binding domain, respectively, in the yeast two-hybridassay.

Crystal Structures of ARA7�VPS9

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Sec7 or Gea2 is supplied into theMg2� binding site and pushesthe Mg2� ion out. DOCK9 uses the side chain of the valineresidue of the nucleotide sensor to occlude the nucleotide coor-dinated Mg2� directly from the Cdc42�GDP�Mg2�, leading todestabilization of GDP. In contrast, the introduction ofGTP�Mg2� into the nucleotide-free Cdc42�DOCK9 complexpushes out the valine side chain, resulting in the release of acti-vated Cdc42 from DOCK9. In this structure, the side chain ofCdc42 switch II Ala59 does not interferewith theMg2� binding,unlike that of Ras Ala59 and Rop Ala62 (6).GDP Destabilization by Aspartate Finger—The exclusion of

Mg2� alone does not seem to be enough to induce the dissoci-ation of GDP from ARA7 small GTPase, although it has beenreported that the loss of Mg2� weakens nucleotide affinity by500–1000 times (40). The trans-type GEFs, such as Vps9 andSec7 domains with the acidic finger, do not have extra motifs,such as the PRONE WW motif stabilizing the nucleotide-freeform or the nucleotide sensor of DOCK proteins responsiblefor the removal of GDP andMg2�. Instead, they accomplish thetask of removing Mg2� and GDP by inserting the acidic fingerinto the nucleotide binding site first to push Mg2� out of thesmall GTPase�GEF complex and then to make contacts withboth GDP �-phosphate and the P-loop lysine. Through thelatter two contacts, VPS9a influences directly the P-loop lysineto move away from GDP toward the acidic residue of switch II(see below). After the GDP release, the acidic finger keeps thecontact with the P-loop lysine to stabilize the nucleotide-freecomplex. This direct GDP recognition by VPS9a, as opposed toindirect GDP recognition by switch II acidic residues, makesthe ARA7�VPS9 GEF mechanism distinct from those of theother small GTPases�GEFs, such as Rop4�PRONE8 andCdc42�DOCK9 complexes.Interaction between P-loop Lysine and Switch II Acidic

Residues—As the third step, it is necessary to move the P-looplysine away from GDP. To do this, an acidic residue, either thefirst aspartate or the last glutamate in the conserved DTAGQEmotif of switch II, attracts the P-loop lysine by an electrostaticinteraction. During GDP release from ARA7�GDP�VPS9, theP-loop lysine swings from the GDP �-phosphate toward switchII Asp65, and as a result, it changes the interaction partner fromthe �-phosphate of GDP to the switch II aspartate. Usually, thelatter interaction between the P-loop lysine and the switch IIacidic residue is formed in the nucleotide-free complexes, asshown in Ras�SOS (38), Rac�Tiam1 (39), or Arf�Gea2 (32), and itis proposed that this coordination is important for keeping thestable nucleotide-free complex stable (40). In the Ran�RCC1complex, the conservedP-loop lysine is detached from the posi-tion nearGDP�-phosphate and is shifted toward the two acidicresidues, Glu70 and Asp65 (Glu62 and Asp57 in Ras) (41). Thisglutamate residue is crucial for the Ran�RCC1 and Ras�Cdc25reactions and is conserved in most Ras-like small GTPases.Interestingly, the comparison of the GDP-bound (6) and

nucleotide-free (42) complex structures of the Rops�PRONE8shows that there is no overall structural change between thesetwo forms and that the electrostatic interaction between ROP4P-loop Lys19 and the �-phosphate oxygen of GDP is alreadyformed in theGDP-bound state (3.7Å inACchains and 3.3Å inBD chains in the ROP4�GDP�PRONE8, and 2.6 Å and 3.3 Å in

the nucleotide-free ROP7�PRONE8, respectively). These obser-vationsmight suggest that the structural rearrangements of thenucleotide binding site necessary for the GDP release werealready accomplished in the GDP-bound state of theROP4�GDP�PRONE8 complex. Also, the structure of the nucle-otide-free ROP7�PRONE8 complex shows that the interactionbetween the ROP7 P-loop Lys19 and the switch II Glu65 isimportant but not sufficient for the stabilization of the nucle-otide-free complex requiring assistance of the WWmotif.In our case, ARA7 Glu70, which corresponds to the con-

served glutamate of switch II of Rop4/7, Ras, or Ran, is furtheraway from the GDP, and the distance between the NH3

� nitro-gen of Lys23 and theCOO� oxygen ofGlu70 is 12Å. Thus, Glu70of ARA7 switch II cannot play the same role as Glu65 of Rop4/7.Instead, Asp65 of ARA7 switch II is situated near the P-looplysine and plays the same role as Rop4Glu65 to attract the lysineNH3

� to stabilize the nucleotide-free formofARA7�VPS9a. Thisis similar to the case of the Cdc42�GDP�DOCK9 structure,where the side chain of Cdc42 P-loop Lys16 is stabilized by theinteraction with Cdc42 Asp57, not Glu62, of switch II (7).Possibility of ARA7�GTP�VPS9a Complex—Finally, we con-

sider if it would be possible for the ARA7�VPS9a complexto accommodate GTP without disintegrating into ARA7and VPS9a. Superimposing the ARA7�VPS9a complex ontoRab5A�GppNHp�Mg2� indicated that the phosphorus atom ofthe �-phosphate would suffer severe steric hindrance with theOD2 of VPS9a Asp185 at a distance of 0.5 Å (Fig. 4a). The side-chain COO� of VPS9a Asp185 cannot drift far from the phos-phorus position in the complex of ARA7�GDP�VPS9a becausethe COO� of VPS9a Asp185 interacts rather tightly with theARA7 Lys23 side chain and the main-chain NH group of theARA7Gly68. However, a structural comparison with the crystalstructure of the nucleotide-free Rab21�Rabex-5 complex (43)sheds light on the possibility of GTP binding. Surprisingly, asuperposition of the two Vps9a domains, VPS9a and Rabex-5,reveals a very large rotation by 18° and displacement by 0.4 Åbetween the twoGTPases, ARA7 and Rab21 (supplemental Fig.10a). This seems to create a space large enough for binding ofGTP in Rab21�Rabex-5 without obvious steric clashes. Hence,we constructed a model in which GTP is introduced to thecorresponding position in the nucleotide-free Rab21�Rabex-5structure (supplemental Fig. 10b). Thismodel shows the recog-nition of the GTP �-phosphate by the aspartate finger throughhydrogen bonds, which are shifted from but similar to thoseobserved for the �-phosphate of GDP in the ARA7�GDP�VPS9astructure.Conclusion—It is now emerging that there are two mecha-

nisms for destabilization of GDP in the nucleotide exchangereaction: either by a direct involvement of GEF with its acidicfinger or by an indirect process where GEF assists the switch IIglutamate of GTPase to approachGDP. However, there remainmany critical questions, such as structural evidence for direc-tionality of the GEF reaction (i.e. from GDP to GTP or viceversa) and whether GEF recognizes Mg2�-bound GTPase intheGDP formas the first step in the forward reaction. Themorerecent structure of a complex between Mg2�-bound RhoGTPase and DOCK complex structure (7) provides an exampleof suchMg2� recognition, although it is GTP and not GDP that

Crystal Structures of ARA7�VPS9

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is recognized. These nucleotide-bound and nucleotide-freeCdc42�DOCK9 structures represent snap shots correspondingto steps much further down the GEF reaction. Further crystal-lographic studies on GDP-bound complexes with magnesiumor other divalentmetal ions would lead to amuch better under-standing of the initial reaction pathway of the GEF reaction ofRab5�VPS9a.

Acknowledgments—We thank the Photon Factory staff for encourage-ment, helpful discussions, and assistance in data collection.We thankthe staff at the Stanford Synchrotron Radiation Lightsource andSPring-8 for assistance during data collection.We thankT.Demura ofRIKEN for providing the plasmid pBD-GAL4-GWRFC.

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Crystal Structures of ARA7�VPS9

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Akihiko Nakano and Soichi WakatsukiTamami Uejima, Kentaro Ihara, Tatsuaki Goh, Emi Ito, Mariko Sunada, Takashi Ueda,

Exchange in the Rab5·Vps9 SystemGDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide

doi: 10.1074/jbc.M110.152132 originally published online September 10, 20102010, 285:36689-36697.J. Biol. Chem. 

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