Molecular Chaperone Hsp70/Hsp90 Prepares theMitochondrial Outer Membrane Translocon Receptor Tom71for Preprotein Loading*

Received for publication, May 27, 2009, and in revised form, July 2, 2009 Published, JBC Papers in Press, July 6, 2009, DOI 10.1074/jbc.M109.023986

Jingzhi Li, Xinguo Qian, Junbin Hu, and Bingdong Sha1

From the Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

Thepreproteins targeted to themitochondria are transportedthrough the translocase of the outer membrane complex.Tom70/Tom71 is a major surface receptor of the translocaseof the outer membrane complex for mitochondrial preproteins.The preproteins are escorted to Tom70/Tom71 by molecularchaperones Hsp70 andHsp90. Here we present the high resolu-tion crystal structures of Tom71 and the protein complexesbetween Tom71 and the Hsp70/Hsp90 C terminus. The crystalstructures indicate that Tom70/Tom71may exhibit two distinctstates. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In theopen state, the N-terminal domainmoves away, and the prepro-tein-binding pocket is fully exposed. The complex formationbetween the C-terminal EEVD motif of Hsp70/Hsp90 andTom71 could lock Tom71 in the open state where the prepro-tein-binding pocket of Tom71 is ready to receive preproteins.The interactions betweenHsp70/Hsp90 andTom71N-terminaldomain generate conformational changes that may increase thevolume of the preprotein-binding pocket. The complex forma-tion of Hsp70/Hsp90 and Tom71 also generates significantdomain rearrangement within Tom71, which may position thepreprotein-binding pocket closer to Hsp70/Hsp90 to facilitatethe preprotein transfer from the molecular chaperone toTom71. Therefore, molecular chaperone Hsp70/Hsp90 mayfunction to prepare the mitochondrial outer membrane recep-tor Tom71 for preprotein loading.

The mitochondrion plays important roles in cell physiology.The mitochondrion functions as the “cellular power house” bygenerating most of the supply of ATP for the cell. In addition,the mitochondrion is involved in a number of critical cellularprocesses including the synthesis of metabolites, lipid metabo-lism, free radical production, and metal ion homeostasis. Themitochondrion consists of four compartments, the outermem-brane, the inner membrane, the intermembrane space, and the

mitochondrial matrix. The mitochondrion contains a largenumber of proteins (1), but only a few of these are translatedwithin the mitochondrion (2). Therefore, the majority of themitochondrial proteins are synthesized in the cytosol andtranslocated into the mitochondrion.The mitochondrial preproteins contain specific targeting

signals to reach the correct compartments within the mi-tochondria. The mitochondrial matrix preproteins containN-terminal targeting sequences that form the short am-phipathic helices (2–6). On the other hand, some mitochon-drial proteins of the inner andoutermembrane contain internaltargeting signals within themature proteins (7). Themitochon-drion has developed a set of delicate translocons to transportthe preproteins into the mitochondrial compartments, onetranslocase of the outer membrane (TOM)2 and two translo-cases of the inner membrane (TIM23 and TIM22) (4, 5, 8). TheTOM complex has two surface receptors, Tom20 and Tom70(9, 10). Tom20 recognizes the N-terminal mitochondrial tar-geting signals from the preproteins, whereas Tom70 binds tointernal targeting sequences of preproteins such as the multi-transmembrane carrier proteins residing in the mitochondrialmembranes (9–12). The crystal structure of Saccharomycescerevisiae Tom70 revealed that Tom70 contained 11 TPRmotifs, and the TPR motifs were clustered into two domains.The three TPR motifs in the N-terminal domain of Tom70pform a peptide-binding groove for the C-terminal EEVD motifof Hsp70/Hsp90, whereas the C-terminal domain of Tom70pcontains a large preprotein-binding pocket (13).Molecular chaperones Hsp70 and Hsp90 play important

roles in targeting the preproteins to TOM complex (14). Hsp70and Hsp90 can protect these preproteins from aggregation inthe cytosol (15). The C-terminal EEVDmotifs of Hsp70/Hsp90may interact directlywith theN-terminal domain ofTom70p totarget the preproteins to TOMcomplex (13, 14, 16). The C-ter-minal EEVD motif of Hsp70/Hsp90 has been indicated to bindseveral proteins containing TPR motifs including Hop andCHIP. The complex structures for the Hsp70/Hsp90 EEVDmotif and Hop and CHIP TPR regions have been determined(17–21).Tom71 (also known as Tom72) was identified as a homo-

logue with Tom70 with high amino acid sequence identity(�50%) (22). Tom71 shares overlapping functions with Tom70to transfer the preproteins and maintain the mitochondrialmorphology (23, 24). In this study, we have determined the

* This work was supported, in whole or in part, by National Institutes of HealthGrants R01 DK56203 and R01 GM65959. This work was also supported byArmy Research Office Grant 51894LS.

The atomic coordinates and structure factors (codes 3FP2, 3FP3, and 3FP4)have been deposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence should be addressed: MCLM 364, 1918 UniversityBlvd., Dept. of Cell Biology, University of Alabama at Birmingham, Birming-ham, AL 35294-0005. Tel.: 205-934-6446; Fax: 205-975-5648; E-mail:bdsha@uab.edu. 2 The abbreviations used are: TOM, translocase of the outer membrane .

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 35, pp. 23852–23859, August 28, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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crystal structures of S. cerevisiae Tom71 and the complexes ofTom71 and Hsp70/Hsp90 C-terminal EEVD motifs. Thesestructures suggest that the Hsp70/Hsp90 binding to Tom70/Tom71may keepTom70/Tom71 in the open state for receivingpreproteins. The Hsp70/Hsp90 interactions may also increasethe volume of the preprotein-binding pocket of Tom70/Tom71and prepare Tom70/Tom71 for preprotein loading.

MATERIALS AND METHODS

Expression and Crystallization of Tom71, Tom71-Hsp70, andTom71-Hsp90 Complexes—The gene encoding S. cerevisiaeTom71cytosolic fragment (residues107–639)was cloned into thevector pET28b. The Tom71 sequence was confirmed by DNAsequencing. The plasmid was then transformed into Escherichiacoli strainBL21 (DE3) forprotein expression.TheBL21(DE3) cellswere inducedwith0.2mMisopropyl-�-D-thiogalactopyranosideatA600 of 0.5–0.6. After shaking overnight at 15 °C, the cells wereharvestedand sonicated in abufferwith20mMTris (pH7.9),NaCl500mM, imidazole 5mM.The supernatantwas collected after cen-trifugation at 15,000� g for 1 h and loaded to nickel-nitrilotriace-ticacidaffinitybeads (GEHealthcare).Theproteinwasstrippedbybuffer containing 20 mM Tris (pH 7.9), 500 mM NaCl, 50 mM

EDTA. After thrombin cleavage overnight at room temperature,the protein was concentrated and purified by Superdex 200 (GEHealthcare) in a buffer with 10 mM Tris (pH 7.5), 150 mM NaCl.The peak fractionwas collected, concentrated to 20mgml�1, andsubjected to crystallization trials. Large rod-shaped crystals (0.5�0.1� 0.1mm)were obtained by the hanging drop vapor diffusionmethod at room temperature. The well solution consisted of 1mlof100mMTrisbuffer (pH7.5), 25%(w/v)polyethyleneglycol4000,0.2 M NaCl. The crystals of Tom71 complexed with yeast Hsp70Ssa1 C-terminal peptide (PEAEGPTVEEVD) were grown by thehanging drop vapor diffusion method by mixing Tom71 andHsp70 peptide using a 1:1.2 molar ratio in buffer 10 mM Tris (pH7.5), 100mMNaCl. The well solution contains 1 ml of 100 mM

Tris buffer (pH 7.5), 20% polyethylene glycol 6000, 10% eth-ylene glycol. The crystals of yeast Tom71 and yeast Hsp90Hsp82 C-terminal peptide (EVPADTEMEEVD) complexwere obtained using similar protocols as for the Tom71and Hsp70 complexes. The peptides were synthesized inGenscript.Structure Determinations—The Tom71 crystals diffracted

x-ray to 2.0 Å in the beamline Southeast Regional CollaborativeAccess Team at Advanced Photon Source. The atomic coordi-nates of theN- andC-terminal domainsof yeastTom70wereusedindividually to search formolecular replacement solutions for thecrystals ofTom71by theprogramPHASER (27).The initialmodelwas built by use of WARP/ARP at 2.0 Å resolution (28), followedbymanualmodel building using COOT (29). The finalmodel wasrefined by REFMAC5 to and Rfactor of 18.7% and an Rfree of 22.7%(30). Tom71 complexed with Hsp70 crystals diffracted x-ray to2.15 Å in the beamline Southeast Regional Collaborative AccessTeam at Advanced Photon Source. The refined atomic coordi-nates of Tom71 were used as a searching model to obtain molec-ular replacement solution. The initial model building was com-pleted by use of WARP/ARP at 2.15 Å resolution, followed bymanualmodel building usingCOOT.The finalmodel was refinedby REFMAC5 to an Rfactor of 20.8% and an Rfree of 26.1%. Tom71

complexed with yeast Hsp90 (Hsp82) crystals diffracted x-ray to2.0 Å in the beamline Southeast Regional Collaborative AccessTeam at Advanced Photon Source. The structure of yeast Tom71and yeast Hsp90 (Hsp82) C-terminal peptide complex was solvedusing similar protocols as for Tom71-Hsp70 complex. The com-plex structure was refined with an Rfactor of 19.9% and an Rfree of23.6%.Sequence Conservation Drawing—To generate the sequence

conservation drawing, program ClustalW was utilized to alignthe Tom71 sequences from S. cerevisiae with those from Can-dida albicans andTom70 from S. cerevisiae,Homo sapiens, andDrosophila melanogaster. The aligned sequences in multiplesequence alignment formats were converted into a property fileby use of the program ProSkin. The property file was then visu-alized by using the program Pymol.

RESULTS AND DISCUSSION

Tom71 May Exhibit Open and Closed States—The S. cerevi-siae Tom71 (639 amino acids) contains a short N-terminaltransmembrane fragment anchored in themitochondrial outermembrane and a large C-terminal fragment located in cytosol.The crystal structure of yeast Tom71 (residues 107–639) wasdetermined to 2.0 Å resolution by a molecular replacementmethod using the yeast Tom70 structure as the searchingmodel (Table 1). The structure of the Tom71 monomer con-sists of 28 �-helices (A0–A27) and no �-strands (Fig. 1, a andb). The electron densities for the loops between helices A1 andA2, A7 and A8, and A22 and A23 are missing. Amajority of thehelices of Tom71 form 11 TPR motifs (TPR1–TPR11). Twodomains can be clearly identified from the Tom71 structure.The N-terminal domain covers helices A0–A7 (TPR1–TPR3),and the C-terminal domain includes helices A8–A27 (TPR4–TPR11). The N-terminal domain of Tom71 contains theHsp70/Hsp90-binding site, and the C-terminal domain consti-tutes the preprotein-binding pocket. The two domains are con-nected by a long helix A7. Most of the helices in the C-terminaldomain (A10–A26) stack together to form one turn of the“superhelix” (Fig. 1, a and b).

TABLE 1Data collection and refinement statistics for crystal structures ofyeast Tom71, Tom71-Hsp70 complex, and Tom71-Hsp90 complexStatistics in the highest resolution shell are shown in parentheses.

Tom71 Tom71-Hsp70 Tom71-Hsp90

Data collectionSpace group P212121 P212121 P212121Cell dimensions: a, b, c (Å) 76.90, 83.42,

109.1147.82, 116.03,

150.6547.87, 116.29,

150.74Wavelength (Å) 1.000 1.000 0.9718Resolution (Å) 2.0 2.15 2.0Rsym or Rmerge 0.053 (0.226) 0.078 (0.506) 0.088 (0.446)I/�I 47.1 (9.2) 19.2 (2.3) 25.0 (2.47)Completeness (%) 99.8 (99.9) 90.3 (77.7) 97.2 (86.1)Redundancy 7.2 (7.0) 5.0 (3.9) 5.0 (2.5)

RefinementNo. reflections 47,008 42,316 57,803Rwork/Rfree 18.9 (21.9)/

22.7 (28.3)20.8 (26.1)/26.1 (35.3)

19.9 (24.7)/23.6 (27.9)

No. atoms protein (water) 4735 (573) 4427 (268) 4559 (451)B factors 34.55 17.85 36.59Root mean square deviationsBond lengths (Å) 0.018 0.023 0.018Bond angels (°) 1.46 1.787 1.524

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When the crystal structure of yeast Tom71 is compared withthat of Tom70, we found that the individual N- and C-terminaldomains of Tom71 resemble the folds from those of Tom70

(Fig. 1, a and c) (13). Surprisingly, the N- and C-terminaldomains of Tom71 are arranged in a very different fashion fromthat in the Tom70 crystal structure. In the crystal structure of

FIGURE 1. The yeast Tom71 structure. a, ribbon drawing of the yeast Tom71 monomer structure (open state) in side-by-side stereo mode (31). The N- andC-terminal domains of Tom71 are shown in light blue and green, respectively. The N (N-ter) and C termini (C-ter) are labeled. TPR1–TPR3 and TPR9 –TPR11 arelabeled. The missing residues in the electron density map are shown in dotted lines. b, ribbon drawing of the yeast Tom71 monomer structure in side-by-sidestereo mode. The Tom71 structure in this figure is rotated �90° along the horizontal axis from that in a. The N and C termini of the structure are labeled.TPR1–TPR11 are labeled. c, ribbon drawing of the yeast Tom70 monomer structure (closed state) in side-by-side stereo mode. The coloring of the N- andC-terminal domains of yeast Tom70 is the same as a. d, the sequence conservation drawing for Tom71. Tom71 in this figure is in similar orientation as that ina. The sequence conservation score obtained by sequence alignment (see Fig. 2 for details) is mapped to the Tom71 molecular surface by Pymol. The red colordenotes the conserved regions. The Hsp70/Hsp90-binding site and the preprotein-binding pocket located on the Tom71 surface are indicated by dotted circles.To indicate the size of the preprotein-binding pocket of Tom71, the distance (27 Å) between Pro234 and Phe496 is shown by an arrow. e, the hydrophobicitydrawing of Tom71 monomer by Pymol. Gold color denotes hydrophobic regions. Tom71 in this figure is rotated �90° along the horizontal axis from that in dand is in similar orientation as that in b. The distance (27 Å) between Pro234 and Phe496 is shown by an arrow. The blue box covers the preprotein-binding pocket,which will be amplified in f. f, this figure shows the magnified version of the area within the blue box in the e. Some conserved hydrophobic residues involvedin forming the preprotein-binding pocket are labeled.

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Tom71, the N-terminal domain is swung away from the C-ter-minal domain, which renders the Tom71 structure an “open”state (Fig. 1a). In the yeast Tom70 structure, the two domainsare packed together to form an elongated molecule as a super-helix, which represents a “closed” state (Fig. 1c). The N-termi-nal domain in the Tom71 structure (open state) moves awayfrom that in the Tom70 structure (closed state) along helix A7.A putative preprotein-binding pocket was identified in theTom70 C-terminal domain (13). In the Tom70 structure, thepreprotein-binding pocket is partially blocked by the N-termi-nal domain. However, in the Tom71 structure, the preprotein-binding pocket is dramatically enlarged and fully exposed andaccessible to the solvent (Fig. 1). In the closed state, the N-ter-minal domain of Tom70 is connected to theC-terminal domainby polar interactions betweenA7 andA25. In the open state, theN-terminal domain of Tom71 is associated to the C-terminaldomain by hydrophobic interactions between A6, A7, and A26.Because yeast Tom70 and Tom71 share high protein

sequence homology (53% sequence identity) and have overlap-ping cellular functions, we reason that Tom70 and Tom71mayadopt both open and closed states in solution and on the mito-chondrial membrane. In the closed state, the preprotein-bind-ing pocket of Tom70/Tom71 is blocked and not ready toreceive preprotein, whereas in the open state, the pocket isexposed for preprotein loading. Previous biophysical and ther-modynamics studies showed that Tom70 exhibited substantialstructural flexibility andmay undergomultiple conformationalchanges at physiological temperature (25). This is consistentwith our structural observations.In the open state, the N-terminal domain of Tom71 moves

away, which creates significantly more space for the prepro-tein-binding pocket. In the open state, the pocket has an esti-mated dimension of �25 � 35 � 20 Å (width � length �depth), whichmore thandoubles the size of pocket in the closedstate. The preprotein-binding pocket is large enough to accom-modate a polypeptide with secondary structures. A pair of�-helices can be easily modeled into the pocket. Helices A12,A14, A16, A18, A20, A22, A24, and A26 from the C-terminaldomain of Tim71 constitute the inner surface of the prepro-tein-binding pocket. Helices A6 and A7 from the N-terminaldomain of Tom71 are also involved in forming one side of thewall for the pocket (Fig. 1).The conservation drawing of Tom71 structure showed

that the residues involved in forming the preprotein-bindingpocket surface are quite conserved among Tom70 andTom71 families, indicating that Tom70 and Tom71 proteinsmay share similar specificity for preproteins (Fig. 1d). A largeportion of the conserved areas in the preprotein-bindingpocket are generated by hydrophobic residues. On the upperpart of the pocket, residues Met215, Leu223, Leu236, Leu240,Leu575, Phe606, and Ala609 form a long hydrophobic strip. Onthe lower half of the pocket, residues Pro264, Phe272, Phe356,Leu357, Leu386, Ala389, Phe423, Ile424, Tyr447, and Phe485 formmost of the bottom of the pocket (Fig. 1, e and f). The hydro-phobicitiy of these residues is nicely conserved as shown bysequence alignment (Fig. 2). This structural feature is consist-ent with the hypothesis that Tom70/Tom71 recognizes the

hydrophobic internal targeting sequences from the preproteins(12, 16).The Interactions between Tom71 and Hsp70/Hsp90 C

Terminus—Molecular chaperone Hsp70/Hsp90 can target thepreproteins to Tom70/Tom71 by anchoring the Hsp70/Hsp90C-terminal EEVD motif to the N-terminal domain of Tom70/Tom71 (13, 14). Here we present the crystal structure of yeastTom71 complexed with yeast Hsp70 (Ssa1) C-terminal peptidePEAEGPTVEEVD to 2.15Å resolution. The crystal structure ofyeast Tom71 complexed with yeast Hsp90 (Hsp82) C-terminalpeptide EVPADTEMEEVDwas determined to 2.0Å resolution.Residues PTVEEVD (residues �6 to 0) of Hsp70 and residuesMEEVD (residues �4 to 0) of Hsp90 are clearly visible in theelectron density map. Both bound peptides exhibit similar con-formations and are located in a groove formedwithin theN-ter-minal domain of Tom71 (Fig. 3). TheHsp70/Hsp90 C-terminalpeptide binds to a region of Tom71 that is overall quite basic. Inboth complex structures, the Asp at position 0 of Hsp70/90makes polar interactions with residues Lys131, Asn135, andLys196 from Tom71. The main chain nitrogen of this Asp resi-due forms a hydrogen bond with the side chain of Asn166 ofTom71. The Val at position �1 makes hydrophobic interac-tions with Phe138. The carbonyl oxygen of Glu at position �2forms strong hydrogen bonds with the side chains of Lys196 andArg200 of Tom71. The Val at position �4 of Hsp70 makeshydrophobic interactions with Leu199 of Tom71. The Met atposition�4 ofHsp90 contacts Leu199 and the hydrophobic sidechain of Lys196 from Tom71. These residues (Lys131, Asn135,Phe138, Asn166, Lys196, Leu199, and Arg200) of Tom71 involvedin binding theC-terminal EEVDmotif ofHsp70/Hsp90 are verynicely conserved for Tom71 and Tom70 among species, sug-gesting that the binding mechanism is shared within theTom70/Tom71 family members (Fig. 2). These residues form aconserved surface at the Hsp70/90-binding site located on theN-terminal domain of Tom71 (Fig. 1d).The hydrophobic interactions between the Val/Met at the

�4 position of Hsp70/Hsp90 with Leu199 of Tom71 make thebound Hsp70/Hsp90 peptide to form a turn at the upstream ofthe EEVD fragment of Hsp70/Hsp90 (Fig. 3, b and d). The turnof the bound peptide indicated that the upstream part of theHsp70/Hsp90 C terminus does not make specific contacts withTom70/Tom71. This phenomenon is also observed in the com-plex structure of CHIP and the Hsp90 C-terminal EEVD motif(20). This may provide flexibility for Hsp70/Hsp90 to deliverthe bound preprotein to the Tom70/Tom71 preprotein-bind-ing pocket.The binding of the Hsp70/Hsp90 C-terminal EEVDmotif to

Tom71 may lock Tom71 in the open state. The bound EEVDmotif of Hsp70/Hsp90 may cause severe collisions with a loopregion between A22 and A23 of Tom71 C-terminal domain ifthe N-terminal domain of Tom71 moves back into the closedstate. Tom71 has to stay in the open state after molecular chap-erone Hsp70/Hsp90 binding, which is the favored state forHsp70/Hsp90 to load the preproteins.The Conformational Changes of Tom71Generated byHsp70/

Hsp90 Binding—The binding of the Hsp70/Hsp90 C-termi-nal EEVD motif to Tom71 generates significant conforma-tional changes in the Tom71 structure (Fig. 4).WhenHsp70/

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Hsp90 binds the Tom71 N-terminal domain, Lys196 andArg200 from helix A5 form a salt bridge and strong hydrogenbonds with Asp at position 0 and Glu at position �2 ofHsp70/Hsp90 C terminus. Leu199 of helix A5 forms hydro-phobic interactions with the Val at position �4 of Hsp70 (orMet at the position �4 of Hsp90). All of these interactions

pull helix A5 of Tom71 toward thebound Hsp70/Hsp90 C terminuspeptide by �1.5 Å after Hsp70/Hsp90 binding (Fig. 4a). Helix A5of Tom71 is associated with heli-ces A6 and A7 by a hydrophobiccluster formed by Leu198, Leu199,and Ala202 from A5; Leu218,Leu221, and Val220 from A6; andMet235 from the N terminus of thelong helix A7. The salt bridgesformed by Arg201 from A5, Asp217from A6, Glu206 from A5, andArg238 from A7 also contribute tothe association of helices A5, A6,and A7. The binding between theHsp70/Hsp90 C-terminal EEVDmotifs and Tom71 cause helix A5of Tom71 to move to the boundHsp70/Hsp90 peptide by �1.5 Å.Because of the association of heli-ces A5, A6, and A7, the conforma-tional changes are translated to A6and A7 and, surprisingly, signifi-cantly amplified within helices A6and A7. After Hsp70/Hsp90 bind-ing to Tom71, helix A6 of Tom71moves toward the bound Hsp70/Hsp90 peptide by �2.5 Å. HelixA7 moves toward the center ofN-terminal domain by �4.5 Å.More importantly, the pulling atthe N terminus of helix A7 causesthe long helix A7 to rotate �20°after Hsp70/Hsp90 binding (Fig.4a). The residues of Tom71involved in mediating the confor-mational changes (Lys196, Leu198,Leu199, Arg200, Arg201, Ala202,Glu206, Asp217, Leu218, Val220,Leu221, Met235, and Arg238) arewell conserved among the Tom70/Tom71 family (Fig. 2).The N-terminal domain of

Tom70/Tom71 may function as asensor to detect the presence ofthe molecular chaperone Hsp70/Hsp90. Lys196 andArg200 of Tom71play central roles to bind Hsp70/90and induce the subsequent confor-mational changes. Lys196 andArg200protrude out of the molecular sur-

face of Tom71 to form strong hydrogen bonds and salt bridgeswith the Hsp70/90 C-terminal EEVDmotif (Fig. 3). The inter-actions between Lys196/Arg200 and Hsp70/Hsp90 function asthe major driving force for the downstream conformationalchanges. The functions of the conserved Lys196 and Arg200 canbe defined as a “Lys/Arg switch.”

FIGURE 2. Sequence alignment of the Tom71/Tom70 family members. Program ClustalW was utilized toalign the Tom71 sequences from S. cerevisiae (Sc Tom71) with that from C. albicans (Ca Tom71) and Tom70 fromS. cerevisiae (Sc Tom70), H. sapiens (Hs Tom70), and D. melanogaster (Dm Tom70). The amino acid residues ofyeast Tom71 are numbered above the alignment. The eleven TPR motifs (TPR1–TPR11) within Tom71 arelabeled. Helices A0 –A27 are labeled. The conserved residues involved in binding the Hsp70/Hsp90 C-terminalEEVD motifs are labeled with blue bars. The conserved residues involved in mediating the conformationalchanges generated by Hsp70/Hsp90 binding are marked with pink bars. The conserved hydrophobic residuesforming the Tom70p preprotein-binding pocket among the family are marked with green bars.

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The structural consequence of the Hsp70/Hsp90 EEVDmotif binding to Tom71 is that the volume of the preprotein-binding pocket located on the C-terminal domain of Tom71 issignificantly increased. Because helices A6 and A7 are involvedin forming one side of the preprotein-binding pocket, the trans-lations of helices A6 and A7 toward the center of the N-termi-nal domain after Hsp70/Hsp90 binding widens up the pocketlocated at the C-terminal domain substantially (Fig. 4a). Inaddition, the rotation of helix A7 after EEVDmotif bindingmayalso contribute to the opening up of the pocket. The width ofthe pocket increases to�30Å afterHsp70/Hsp90 binding from�25 Å before binding. For example, residues Pro234 and Phe496are located at the opposite side on the preprotein-bindingpocket of Tom71. The distance between theC� atoms of Pro234

and Phe496 increases to 32 Å after complex formation, whereasthe distance was 27 Å before Hsp70/Hsp90 binding. (Figs. 1dand 3, a and d). An enlarged preprotein-binding pocket mayfacilitate efficient preprotein loading from Hsp70/Hsp90 toTom70/Tom71. As a similar comparison, the cavity inside ofchaperonin GroEL is also significantly increased after GroESbinding (26).Helix A7 is positioned at the joint of Tom71 N-terminal and

C-terminal domains and acts as a hinge to link the two domainstogether. The rotation of helix A7 (�20°) of Tom71 afterHsp70/Hsp90 binding is transduced to the entire C-terminaldomain and generates significant rearrangement of the N-ter-minal and C-terminal domains of Tom71. The C-terminaldomain of Tom71 is swung from the open state to the closed

FIGURE 3. The complex structures of Tom71 and the Hsp70/Hsp90 C-terminal EEVD motif. a, the surface potential drawing of Tom71 complexed withHsp70 C-terminal peptide PTVEEVD. Tom71 is shown in surface potential drawing generated by Pymol and Apbs. Blue and red denote positively and negativelyelectrostatic potentials, respectively. The bound Hsp70 peptide is shown in a rod model. In the rod model, carbon atoms are shown in green, oxygen atoms areshown in red, and nitrogen atoms are shown in blue. To indicate the enlargement of the preprotein-binding pocket of Tom71 after Hsp70 binding, the distance(32 Å) between Pro234 and Phe496 of Tom71 is shown. b, the surface potential drawing of Tom71 N-terminal domain interacting with the Hsp70 C-terminalpeptide PTVEEVD in a rod model. The residues of Tom71 involved in binding Hsp70 are labeled in green, and the residues of Hsp70 peptide PTVEEVD are labeledin black. c, ribbon drawing of Tom71 N-terminal domain complexed with Hsp70 C terminus in stereo mode. Tom71 is in a silver ribbon drawing, and the Hsp70C-terminal peptide is in a solid rod model. The residues of Tom71 involved in binding Hsp70 are drawn in dotted rod model and labeled in blue. The residuesof Hsp70 peptide are labeled in black. d, the surface potential drawing of Tom71 N-terminal domain interacting with Hsp90 C-terminal peptide MEEVD in a rodmodel. The residues of Tom71 involved in binding Hsp90 are labeled in green, and the residues of Hsp90 peptide MEEVD are labeled in black.

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state by �20° after the Hsp70/Hsp90 binding (Fig. 4a). The 20°rotation of the Tom71 C-terminal domain after the Hsp70/Hsp90 binding can bring the preprotein-binding pocket muchcloser to the molecular chaperone Hsp70/Hsp90, which mayfacilitate the preprotein transfer from Hsp70/Hsp90 to Tom71(Fig. 4b). Therefore the domain rearrangement within Tom71molecule may have significant functional benefit for Tom71 toreceive the preproteins from Hsp70/Hsp90.The crystal structures of yeast Tom71 and Tom70 suggest

that the mitochondrial translocon receptor Tom70/Tom71may exhibit the open and closed states. In the closed state, theN-terminal domain of Tom70/Tom71 folds on top of theC-ter-minal domain and partially covers the preprotein-bindingpocket. This may block the preproteins from entering the

pocket. In the open state, the N-terminal domain moves away,and the preprotein-binding pocket is dramatically enlarged andexposed. When molecular chaperone Hsp70/Hsp90 escortspreproteins to approach the TOM complex, the Hsp70/Hsp90C-terminal EEVD motif will bind the Tom71 N-terminaldomain. The interactions will lock Tom71 in the open state.The binding betweenHsp70/Hsp90 andTom71 could generatesignificant conformational changes in Tom71 structure, whichcould further increase the volume of the preprotein-bindingpocket and rotate the pocket closer to Hsp70/Hsp90. Thus, themolecular chaperone Hsp70/Hsp90 could ensure that Tom70/Tom71 only receive the preproteins escorted by molecularchaperones (Fig. 5). The cooperativity of Tom70/Tom71 andthemolecular chaperone Hsp70/Hsp90 provides a delicate gat-

FIGURE 4. The conformational changes of Tom71 generated by Hsp70binding. a, the N-terminal domain of Tom71 is superimposed with that in theTom71-Hsp70 complex and they are shown by a ribbon drawing. The Tom71N-terminal domain is in light blue. The Tom71 N-terminal domain within theTom71-Hsp70 complex is in silver. The bound Hsp70 C-terminal peptide is inred. Helices A5, A6, and A7 are labeled in blue. Some residues of Tom71involved in generating the conformational changes are labeled in black. Res-idues Lys196, Arg200, and Leu199 of Tom71 involved in binding Hsp70 arelabeled. The residues forming hydrophobic cluster to associate A5, A6, and A7are labeled. Glu206 and Arg238 linking A5 and A7 by forming a salt bridge arealso labeled. b, C� trace drawings of yeast Tom71 structure and the Tom71-Hsp70 complex structure. The N-terminal domain of Tom71 is superimposedwith that in the Tom71-Hsp70 complex structure. The molecules in this figureare in a similar orientation as in a. The uncomplexed Tom71 structure is inpurple. In the Tom71-Hsp70 C terminus complex, Tom71 is in green, and theHsp70 C terminus is in red. The N- and C-terminal domains of Tom71 arelabeled. Helix A7 acting as the hinge to connect the N-and C-terminaldomains of Tom71 is labeled.

FIGURE 5. The cartoon drawing for the mechanism how Hsp70/Hsp90prepares Tom70/Tom71 for preprotein loading. a, Tom70/Tom71 mayexhibit two distinct states: the open and closed state. The Tom70/Tom71molecule is shown in blue. The N- and C-terminal domains are labeled. Themitochondria outer membrane is shown in orange. b, the interactionsbetween the Hsp70/Hsp90 C-terminal EEVD motif will lock the Tom70/Tom71in the open state. The Hsp70/Hsp90 is shown in gold. The binding betweenHsp70/Hsp90 and Tom71 could increase the volume of the preprotein-bind-ing pocket. The complex formation might rotate the Tom71 C-terminaldomain �20° back toward the closed state and therefore position the prepro-tein-binding pocket closer to the Hsp70/Hsp90. The Hsp70/Hsp90 EEVD motifis shown as a red arrow. The preprotein is shown as a green triangle. c, Hsp70/Hsp90 will then load the preprotein into the enlarged preprotein-bindingpocket of Tom70/Tom71.

Hsp70/Hsp90 Prepares Tom71 for Preprotein Loading

23858 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 35 • AUGUST 28, 2009

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ing system for Tom70/Tom71 to recognize the correct prepro-tein substrates.

Acknowledgments—We are grateful to the staff scientists in AdvancedPhoton Source beamline Southeast Regional Collaborative AccessTeam for help with data collection.

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Hsp70/Hsp90 Prepares Tom71 for Preprotein Loading

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Jingzhi Li, Xinguo Qian, Junbin Hu and Bingdong ShaTranslocon Receptor Tom71 for Preprotein Loading

Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane

doi: 10.1074/jbc.M109.023986 originally published online July 6, 20092009, 284:23852-23859.J. Biol. Chem. 

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