Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

Abstract

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading. The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading. The mitochondrion plays important roles in cell physiology. The mitochondrion functions as the “cellular power house” by generating most of the supply of ATP for the cell. In addition, the mitochondrion is involved in a number of critical cellular processes including the synthesis of metabolites, lipid metabolism, free radical production, and metal ion homeostasis. The mitochondrion consists of four compartments, the outer membrane, the inner membrane, the intermembrane space, and the mitochondrial matrix. The mitochondrion contains a large number of proteins (1.Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schönfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13207-13212Crossref PubMed Scopus (718) Google Scholar), but only a few of these are translated within the mitochondrion (2.Gray M.W. Burger G. Lang B.F. Science. 1999; 283: 1476-1481Crossref PubMed Scopus (1379) Google Scholar). Therefore, the majority of the mitochondrial proteins are synthesized in the cytosol and translocated into the mitochondrion. The mitochondrial preproteins contain specific targeting signals to reach the correct compartments within the mitochondria. The mitochondrial matrix preproteins contain N-terminal targeting sequences that form the short amphipathic helices (2.Gray M.W. Burger G. Lang B.F. Science. 1999; 283: 1476-1481Crossref PubMed Scopus (1379) Google Scholar, 3.Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar, 4.Neupert W. Brunner M. Nat. Rev. Mol. Cell Biol. 2002; 3: 555-565Crossref PubMed Scopus (301) Google Scholar, 5.Pfanner N. Curr. Biol. 2000; 10: R412-415Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 6.Abe Y. Shodai T. Muto T. Mihara K. Torii H. Nishikawa S. Endo T. Kohda D. Cell. 2000; 100: 551-560Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). On the other hand, some mitochondrial proteins of the inner and outer membrane contain internal targeting signals within the mature proteins (7.Rehling P. Model K. Brandner K. Kovermann P. Sickmann A. Meyer H.E. Kühlbrandt W. Wagner R. Truscott K.N. Pfanner N. Science. 2003; 299: 1747-1751Crossref PubMed Scopus (234) Google Scholar). The mitochondrion has developed a set of delicate translocons to transport the preproteins into the mitochondrial compartments, one translocase of the outer membrane (TOM) 2The abbreviations used are: TOMtranslocase of the outer membrane. and two translocases of the inner membrane (TIM23 and TIM22) (4.Neupert W. Brunner M. Nat. Rev. Mol. Cell Biol. 2002; 3: 555-565Crossref PubMed Scopus (301) Google Scholar, 5.Pfanner N. Curr. Biol. 2000; 10: R412-415Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8.Koehler C.M. Annu. Rev. Cell Dev. Biol. 2004; 20: 309-335Crossref PubMed Scopus (252) Google Scholar). The TOM complex has two surface receptors, Tom20 and Tom70 (9.Söllner T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 10.Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar). Tom20 recognizes the N-terminal mitochondrial targeting signals from the preproteins, whereas Tom70 binds to internal targeting sequences of preproteins such as the multi-transmembrane carrier proteins residing in the mitochondrial membranes (9.Söllner T. Pfaller R. Griffiths G. Pfanner N. Neupert W. Cell. 1990; 62: 107-115Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 10.Söllner T. Griffiths G. Pfaller R. Pfanner N. Neupert W. Cell. 1989; 59: 1061-1070Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 11.Brix J. Dietmeier K. Pfanner N. J. Biol. Chem. 1997; 272: 20730-20735Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 12.Chan N.C. Likiæ V.A. Waller R.F. Mulhern T.D. Lithgow T. J. Mol. Biol. 2006; 358: 1010-1022Crossref PubMed Scopus (82) Google Scholar). The crystal structure of Saccharomyces cerevisiae Tom70 revealed that Tom70 contained 11 TPR motifs, and the TPR motifs were clustered into two domains. The three TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70/Hsp90, whereas the C-terminal domain of Tom70p contains a large preprotein-binding pocket (13.Wu Y. Sha B. Nat. Struct. Mol. Biol. 2006; 13: 589-593Crossref PubMed Scopus (112) Google Scholar). translocase of the outer membrane. Molecular chaperones Hsp70 and Hsp90 play important roles in targeting the preproteins to TOM complex (14.Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). Hsp70 and Hsp90 can protect these preproteins from aggregation in the cytosol (15.Beddoe T. Lithgow T. Biochim. Biophys. Acta. 2002; 1592: 35-39Crossref PubMed Scopus (53) Google Scholar). The C-terminal EEVD motifs of Hsp70/Hsp90 may interact directly with the N-terminal domain of Tom70p to target the preproteins to TOM complex (13.Wu Y. Sha B. Nat. Struct. Mol. Biol. 2006; 13: 589-593Crossref PubMed Scopus (112) Google Scholar, 14.Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 16.Brix J. Ziegler G.A. Dietmeier K. Schneider-Mergener J. Schulz G.E. Pfanner N. J. Mol. Biol. 2000; 303: 479-488Crossref PubMed Scopus (69) Google Scholar). The C-terminal EEVD motif of Hsp70/Hsp90 has been indicated to bind several proteins containing TPR motifs including Hop and CHIP. The complex structures for the Hsp70/Hsp90 EEVD motif and Hop and CHIP TPR regions have been determined (17.Demand J. Lüders J. Höhfeld J. Mol. Cell. Biol. 1998; 18: 2023-2028Crossref PubMed Scopus (232) Google Scholar, 18.Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell. Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (757) Google Scholar, 19.Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar, 20.Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 21.Scheufler C. Brinker A. Bourenkov G. Pegoraro S. Moroder L. Bartunik H. Hartl F.U. Moarefi I. Cell. 2000; 101: 199-210Abstract Full Text Full Text PDF PubMed Scopus (1016) Google Scholar). Tom71 (also known as Tom72) was identified as a homologue with Tom70 with high amino acid sequence identity (>50%) (22.Bömer U. Pfanner N. Dietmeier K. FEBS Lett. 1996; 382: 153-158Crossref PubMed Scopus (49) Google Scholar). Tom71 shares overlapping functions with Tom70 to transfer the preproteins and maintain the mitochondrial morphology (23.Koh J.Y. Hájek P. Bedwell D.M. Mol. Cell. Biol. 2001; 21: 7576-7586Crossref PubMed Scopus (14) Google Scholar, 24.Kondo-Okamoto N. Shaw J.M. Okamoto K. EMBO Rep. 2008; 9: 63-69Crossref PubMed Scopus (27) Google Scholar). In this study, we have determined the crystal structures of S. cerevisiae Tom71 and the complexes of Tom71 and Hsp70/Hsp90 C-terminal EEVD motifs. These structures suggest that the Hsp70/Hsp90 binding to Tom70/Tom71 may keep Tom70/Tom71 in the open state for receiving preproteins. The Hsp70/Hsp90 interactions may also increase the volume of the preprotein-binding pocket of Tom70/Tom71 and prepare Tom70/Tom71 for preprotein loading. The gene encoding S. cerevisiae Tom71 cytosolic fragment (residues 107–639) was cloned into the vector pET28b. The Tom71 sequence was confirmed by DNA sequencing. The plasmid was then transformed into Escherichia coli strain BL21 (DE3) for protein expression. The BL21(DE3) cells were induced with 0.2 mm isopropyl-β-d-thiogalactopyranoside at A600 of 0.5–0.6. After shaking overnight at 15 °C, the cells were harvested and sonicated in a buffer with 20 mm Tris (pH 7.9), NaCl 500 mm, imidazole 5 mm. The supernatant was collected after centrifugation at 15,000 × g for 1 h and loaded to nickel-nitrilotriacetic acid affinity beads (GE Healthcare). The protein was stripped by buffer 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 (GE Healthcare) in a buffer with 10 mm Tris (pH 7.5), 150 mm NaCl. The peak fraction was collected, concentrated to 20 mg ml−1, and subjected to crystallization trials. Large rod-shaped crystals (0.5 × 0.1 × 0.1 mm) were obtained by the hanging drop vapor diffusion method at room temperature. The well solution consisted of 1 ml of 100 mm Tris buffer (pH 7.5), 25% (w/v) polyethylene glycol 4000, 0.2 m NaCl. The crystals of Tom71 complexed with yeast Hsp70 Ssa1 C-terminal peptide (PEAEGPTVEEVD) were grown by the hanging drop vapor diffusion method by mixing Tom71 and Hsp70 peptide using a 1:1.2 molar ratio in buffer 10 mm Tris (pH 7.5), 100 mm NaCl. The well solution contains 1 ml of 100 mm Tris buffer (pH 7.5), 20% polyethylene glycol 6000, 10% ethylene glycol. The crystals of yeast Tom71 and yeast Hsp90 Hsp82 C-terminal peptide (EVPADTEMEEVD) complex were obtained using similar protocols as for the Tom71 and Hsp70 complexes. The peptides were synthesized in Genscript. The Tom71 crystals diffracted x-ray to 2.0 Å in the beamline Southeast Regional Collaborative Access Team at Advanced Photon Source. The atomic coordinates of the N- and C-terminal domains of yeast Tom70 were used individually to search for molecular replacement solutions for the crystals of Tom71 by the program PHASER (27.CCP4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The initial model was built by use of WARP/ARP at 2.0 Å resolution (28.Langer G. Cohen S.X. Lamzin V.S. Perrakis A. Nat. Prot. 2008; 3: 1171-1179Crossref PubMed Scopus (1332) Google Scholar), followed by manual model building using COOT (29.Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). The final model was refined by REFMAC5 to and Rfactor of 18.7% and an Rfree of 22.7% (30.Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1654) Google Scholar). Tom71 complexed with Hsp70 crystals diffracted x-ray to 2.15 Å in the beamline Southeast Regional Collaborative Access Team at Advanced Photon Source. The refined atomic coordinates of Tom71 were used as a searching model to obtain molecular replacement solution. The initial model building was completed by use of WARP/ARP at 2.15 Å resolution, followed by manual model building using COOT. The final model was refined by REFMAC5 to an Rfactor of 20.8% and an Rfree of 26.1%. Tom71 complexed with yeast Hsp90 (Hsp82) crystals diffracted x-ray to 2.0 Å in the beamline Southeast Regional Collaborative Access Team at Advanced Photon Source. The structure of yeast Tom71 and yeast Hsp90 (Hsp82) C-terminal peptide complex was solved using similar protocols as for Tom71-Hsp70 complex. The complex structure was refined with an Rfactor of 19.9% and an Rfree of 23.6%. To generate the sequence conservation drawing, program ClustalW was utilized to align the Tom71 sequences from S. cerevisiae with those from Candida albicans and Tom70 from S. cerevisiae, Homo sapiens, and Drosophila melanogaster. The aligned sequences in multiple sequence alignment formats were converted into a property file by use of the program ProSkin. The property file was then visualized by using the program Pymol. The S. cerevisiae Tom71 (639 amino acids) contains a short N-terminal transmembrane fragment anchored in the mitochondrial outer membrane and a large C-terminal fragment located in cytosol. The crystal structure of yeast Tom71 (residues 107–639) was determined to 2.0 Å resolution by a molecular replacement method using the yeast Tom70 structure as the searching model (Table 1). The structure of the Tom71 monomer consists of 28 α-helices (A0–A27) and no β-strands (Fig. 1, a and b). The electron densities for the loops between helices A1 and A2, A7 and A8, and A22 and A23 are missing. A majority of the helices of Tom71 form 11 TPR motifs (TPR1–TPR11). Two domains 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 the Hsp70/Hsp90-binding site, and the C-terminal domain constitutes the preprotein-binding pocket. The two domains are connected by a long helix of the helices in the C-terminal domain to form one of the (Fig. 1, a and and for crystal structures of yeast Tom71, Tom71-Hsp70 and protein in a the crystal structure of yeast Tom71 is with that of we that the N- and C-terminal domains of Tom71 the from those of Tom70 (Fig. 1, a and (13.Wu Y. Sha B. Nat. Struct. Mol. Biol. 2006; 13: 589-593Crossref PubMed Scopus (112) Google Scholar). the N- and C-terminal domains of Tom71 are in a from that in the Tom70 crystal structure. In the crystal structure of Tom71, the N-terminal domain is from the C-terminal which the Tom71 structure an state (Fig. In the yeast Tom70 the two domains are to form an as a which a state (Fig. The N-terminal domain in the Tom71 structure moves from that in the Tom70 structure helix A preprotein-binding pocket was identified in the Tom70 C-terminal domain (13.Wu Y. Sha B. Nat. Struct. Mol. Biol. 2006; 13: 589-593Crossref PubMed Scopus (112) Google Scholar). In the Tom70 the preprotein-binding pocket is partially by the N-terminal in the Tom71 the preprotein-binding pocket is and fully and to the (Fig. 1). In the closed state, the N-terminal domain of Tom70 is connected to the C-terminal domain by interactions between A7 and In the open state, the N-terminal domain of Tom71 is to the C-terminal domain by interactions between and yeast Tom70 and Tom71 high protein sequence sequence and have overlapping cellular we that Tom70 and Tom71 may open and closed in solution and the mitochondrial membrane. In the closed state, the preprotein-binding pocket of Tom70/Tom71 is and ready to receive whereas in the open state, the pocket is for preprotein loading. and that Tom70 and may multiple conformational changes at T. Lithgow T. Mulhern T.D. J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). is with In the open state, the N-terminal domain of Tom71 moves away, which for the preprotein-binding pocket. In the open state, the pocket has an of × × 20 Å × × which the of pocket in the closed The preprotein-binding pocket is large to a with A of α-helices can be into the pocket. and from the C-terminal domain of the inner surface of the preprotein-binding pocket. and A7 from the N-terminal domain of Tom71 are also involved in one of the for the pocket (Fig. 1). The conservation of Tom71 structure that the involved in the preprotein-binding pocket surface are Tom70 and Tom71 that Tom70 and Tom71 proteins may similar for preproteins (Fig. A large of the in the preprotein-binding pocket are by On the of the and form a long On the of the and form most of the of the pocket (Fig. 1, and The of these is as by sequence alignment (Fig. is with the that Tom70/Tom71 recognizes the internal targeting sequences from the preproteins N.C. Likiæ V.A. Waller R.F. Mulhern T.D. Lithgow T. J. Mol. Biol. 2006; 358: 1010-1022Crossref PubMed Scopus (82) Google Scholar, 16.Brix J. Ziegler G.A. Dietmeier K. Schneider-Mergener J. Schulz G.E. Pfanner N. J. Mol. Biol. 2000; 303: 479-488Crossref PubMed Scopus (69) Google Scholar). Molecular chaperone Hsp70/Hsp90 can target the preproteins to Tom70/Tom71 by the Hsp70/Hsp90 C-terminal EEVD motif to the N-terminal domain of Tom70/Tom71 (13.Wu Y. Sha B. Nat. Struct. Mol. Biol. 2006; 13: 589-593Crossref PubMed Scopus (112) Google Scholar, 14.Young J.C. Hoogenraad N.J. Hartl F.U. Cell. 2003; 112: 41-50Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). Here we present the crystal structure of yeast Tom71 complexed with yeast Hsp70 C-terminal peptide to 2.15 Å The crystal structure of yeast Tom71 complexed with yeast Hsp90 (Hsp82) C-terminal peptide was determined to 2.0 Å (residues to of Hsp70 and (residues to of Hsp90 are clearly in the electron peptides exhibit similar and are located in a groove within the N-terminal domain of Tom71 (Fig. The Hsp70/Hsp90 C-terminal peptide binds to a of Tom71 that is In complex the at position of interactions with and from Tom71. The of this a with the of of Tom71. The at position interactions with The of at position with the of and of Tom71. The at position of Hsp70 interactions with of Tom71. The at position of Hsp90 and the of from Tom71. These and of Tom71 involved in binding the C-terminal EEVD motif of Hsp70/Hsp90 are for Tom71 and Tom70 that the binding is within the Tom70/Tom71 (Fig. These form a surface at the located the N-terminal domain of Tom71 (Fig. The interactions between the at the position of Hsp70/Hsp90 with of Tom71 the Hsp70/Hsp90 peptide to form a at the of the EEVD fragment of Hsp70/Hsp90 (Fig. and The of the peptide indicated that the of the Hsp70/Hsp90 C specific with is also in the complex structure of CHIP and the Hsp90 C-terminal EEVD motif M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). may for Hsp70/Hsp90 to the preprotein to the Tom70/Tom71 preprotein-binding pocket. The binding of the Hsp70/Hsp90 C-terminal EEVD motif to Tom71 may lock Tom71 in the open The EEVD motif of Hsp70/Hsp90 may with a between A22 and A23 of Tom71 C-terminal domain the N-terminal domain of Tom71 moves into the closed Tom71 has to in the open state after molecular chaperone Hsp70/Hsp90 which is the state for Hsp70/Hsp90 to the preproteins. The binding of the Hsp70/Hsp90 C-terminal EEVD motif to Tom71 generates significant conformational changes in the Tom71 structure (Fig. Hsp70/Hsp90 binds the Tom71 N-terminal and from helix form a and with at position and at position of Hsp70/Hsp90 C terminus. of helix interactions with the at position of Hsp70 at the position of of these interactions helix of Tom71 the Hsp70/Hsp90 C peptide by Å after Hsp70/Hsp90 binding (Fig. of Tom71 is with helices and A7 by a by and from and from and from the of the long helix The by from from from and from A7 also to the of helices and The binding between the Hsp70/Hsp90 C-terminal EEVD motifs and Tom71 helix of Tom71 to to the Hsp70/Hsp90 peptide by of the of helices and the conformational changes are translated to and A7 within helices and After Hsp70/Hsp90 binding to Tom71, helix of Tom71 moves the Hsp70/Hsp90 peptide by A7 moves the of N-terminal domain by the at the of helix A7 the long helix A7 to after Hsp70/Hsp90 binding (Fig. The of Tom71 involved in the conformational changes and are well the Tom70/Tom71 (Fig. The N-terminal domain of Tom70/Tom71 may function as a to the of the molecular chaperone and of Tom71 play roles to bind and the conformational and of the molecular surface of Tom71 to form and with the C-terminal EEVD motif (Fig. The interactions between and Hsp70/Hsp90 function as the major for the conformational The functions of the and can be as a The of the Hsp70/Hsp90 EEVD motif binding to Tom71 is that the volume of the preprotein-binding pocket located the C-terminal domain of Tom71 is helices and A7 are involved in one of the preprotein-binding the of helices and A7 the of the N-terminal domain after Hsp70/Hsp90 binding the pocket located at the C-terminal domain (Fig. In addition, the of helix A7 after EEVD motif binding may also to the of the pocket. The of the pocket to Å after Hsp70/Hsp90 binding from Å and are located at the the preprotein-binding pocket of Tom71. The between the of and to Å after complex whereas the was Å Hsp70/Hsp90 and a and preprotein-binding pocket may facilitate preprotein from Hsp70/Hsp90 to a similar the of is also after binding L. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A7 is at the of Tom71 N-terminal and C-terminal domains and as a to the two domains The of helix A7 of Tom71 after Hsp70/Hsp90 binding is to the C-terminal domain and generates significant rearrangement of the N-terminal and C-terminal domains of Tom71. The C-terminal domain of Tom71 is from the open state to the closed state by after the Hsp70/Hsp90 binding (Fig. The of the Tom71 C-terminal domain after the Hsp70/Hsp90 binding can the preprotein-binding pocket closer to the molecular chaperone Hsp70/Hsp90, which may facilitate the preprotein transfer from Hsp70/Hsp90 to Tom71 (Fig. the domain rearrangement within Tom71 may have significant for Tom71 to receive the preproteins from The crystal structures of yeast Tom71 and Tom70 suggest that the mitochondrial receptor Tom70/Tom71 may exhibit the open and closed states. In the closed state, the N-terminal domain of Tom70/Tom71 of the C-terminal domain and partially covers the preprotein-binding pocket. may the preproteins from the pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is and exposed. molecular chaperone Hsp70/Hsp90 preproteins to the TOM the Hsp70/Hsp90 C-terminal EEVD motif bind the Tom71 N-terminal The interactions lock Tom71 in the open The binding between Hsp70/Hsp90 and Tom71 could generate significant conformational changes in Tom71 which could increase the volume of the preprotein-binding pocket and the pocket closer to the molecular chaperone Hsp70/Hsp90 could that Tom70/Tom71 only receive the preproteins escorted by molecular chaperones (Fig. The of Tom70/Tom71 and the molecular chaperone Hsp70/Hsp90 a delicate for Tom70/Tom71 to the correct preprotein are to the in Advanced Photon beamline Southeast Regional Collaborative Access Team for with