Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

From the Masaryk Memorial Cancer Institute Regional Centre for Applied Molecular Oncology Zluty kopec 7 656 53 Brno Czech Republic

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Krukenberg K. A., Street T. O., Lavery L. A., Agard D. A. (2011) Conformational dynamics of the molecular chaperone Hsp90. Q. Rev. Biophys. 44, 229–255 PubMed PMC

Koulov A. V., LaPointe P., Lu B., Razvi A., Coppinger J., Dong M. Q., Matteson J., Laister R., Arrowsmith C., Yates J. R., 3rd, Balch W. E. (2010) Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis. Mol. Biol. Cell 21, 871–884 PubMed PMC

Takayama S., Bimston D. N., Matsuzawa S., Freeman B. C., Aime-Sempe C., Xie Z., Morimoto R. I., Reed J. C. (1997) BAG-1 modulates the chaperone activity of Hsp70/Hsc70. EMBO J. 16, 4887–4896 PubMed PMC

Allan R. K., Ratajczak T. (2011) Versatile TPR domains accommodate different modes of target protein recognition and function. Cell Stress Chaperones 16, 353–367 PubMed PMC

Cliff M. J., Harris R., Barford D., Ladbury J. E., Williams M. A. (2006) Conformational diversity in the TPR domain-mediated interaction of protein phosphatase 5 with Hsp90. Structure 14, 415–426 PubMed

Cheung-Flynn J., Roberts P. J., Riggs D. L., Smith D. F. (2003) C-terminal sequences outside the tetratricopeptide repeat domain of FKBP51 and FKBP52 cause differential binding to Hsp90. J. Biol. Chem. 278, 17388–17394 PubMed

Scheufler C., Brinker A., Bourenkov G., Pegoraro S., Moroder L., Bartunik H., Hartl F. U., Moarefi I. (2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101, 199–210 PubMed

Lamb J. R., Tugendreich S., Hieter P. (1995) Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem. Sci. 20, 257–259 PubMed

Brinker A., Scheufler C., Von Der Mulbe F., Fleckenstein B., Herrmann C., Jung G., Moarefi I., Hartl F. U. (2002) Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70·Hop·Hsp90 complexes. J. Biol. Chem. 277, 19265–19275 PubMed

Kajander T., Sachs J. N., Goldman A., Regan L. (2009) Electrostatic interactions of Hsp-organizing protein tetratricopeptide domains with Hsp70 and Hsp90: computational analysis and protein engineering. J. Biol. Chem. 284, 25364–25374 PubMed PMC

Ward B. K., Allan R. K., Mok D., Temple S. E., Taylor P., Dornan J., Mark P. J., Shaw D. J., Kumar P., Walkinshaw M. D., Ratajczak T. (2002) A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90. J. Biol. Chem. 277, 40799–40809 PubMed

Schmid A. B., Lagleder S., Gräwert M. A., Röhl A., Hagn F., Wandinger S. K., Cox M. B., Demmer O., Richter K., Groll M., Kessler H., Buchner J. (2012) The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop. EMBO J. 31, 1506–1517 PubMed PMC

Carrigan P. E., Nelson G. M., Roberts P. J., Stoffer J., Riggs D. L., Smith D. F. (2004) Multiple domains of the co-chaperone Hop are important for Hsp70 binding. J. Biol. Chem. 279, 16185–16193 PubMed

Chewawiwat N., Yano M., Terada K., Hoogenraad N. J., Mori M. (1999) Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells. J. Biochem. 125, 721–727 PubMed

Nuttall S. D., Hanson B. J., Mori M., Hoogenraad N. J. (1997) hTom34: a novel translocase for the import of proteins into human mitochondria. DNA Cell Biol. 16, 1067–1074 PubMed

Faou P., Hoogenraad N. J. (2012) Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import. Biochim. Biophys. Acta 1823, 348–357 PubMed

Tropea J. E., Cherry S., Waugh D. S. (2009) Expression and purification of soluble His6-tagged TEV protease. Methods Mol. Biol. 498, 297–307 PubMed

Glover D. M. (1986) DNA Cloning: A Practical Approach, Volume I, II 435 S., 76 Abb., 116 Tab. Oxford-Washington 1985 IRL Press

Kavan D., Man P. (2011) MSTools: Web based application for visualization and presentation of HXMS data. Int. J. Mass Spectrom. 302, 53–58

Karpenahalli M. R., Lupas A. N., Söding J. (2007) TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences. BMC Bioinformatics 8, 2. PubMed PMC

Notredame C., Higgins D. G., Heringa J. (2000) T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 PubMed

Tsaytler P. A., Krijgsveld J., Goerdayal S. S., Rüdiger S., Egmond M. R. (2009) Novel Hsp90 partners discovered using complementary proteomic approaches. Cell Stress Chaperones 14, 629–638 PubMed PMC

Young J. C., Obermann W. M., Hartl F. U. (1998) Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90. J. Biol. Chem. 273, 18007–18010 PubMed

Wayne N., Bolon D. N. (2007) Dimerization of Hsp90 is required for in vivo function. Design and analysis of monomers and dimers. J. Biol. Chem. 282, 35386–35395 PubMed

Aprile F. A., Dhulesia A., Stengel F., Roodveldt C., Benesch J. L., Tortora P., Robinson C. V., Salvatella X., Dobson C. M., Cremades N. (2013) Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain. PLoS One 8, e67961. PubMed PMC

Lee C. T., Graf C., Mayer F. J., Richter S. M., Mayer M. P. (2012) Dynamics of the regulation of Hsp90 by the co-chaperone Sti1. EMBO J. 31, 1518–1528 PubMed PMC

Rezabkova L., Man P., Novak P., Herman P., Vecer J., Obsilova V., Obsil T. (2011) Structural basis for the 14-3-3 protein-dependent inhibition of the regulator of G protein signaling 3 (RGS3) function. J. Biol. Chem. 286, 43527–43536 PubMed PMC

Li J., Buchner J. (2013) Structure, function and regulation of the hsp90 machinery. Biomed. J. 36, 106–117 PubMed

Retzlaff M., Hagn F., Mitschke L., Hessling M., Gugel F., Kessler H., Richter K., Buchner J. (2010) Asymmetric activation of the hsp90 dimer by its cochaperone aha1. Mol. Cell 37, 344–354 PubMed

Sun L., Prince T., Manjarrez J. R., Scroggins B. T., Matts R. L. (2012) Characterization of the interaction of Aha1 with components of the Hsp90 chaperone machine and client proteins. Biochim. Biophys. Acta 1823, 1092–1101 PubMed

Stebbins C. E., Russo A. A., Schneider C., Rosen N., Hartl F. U., Pavletich N. P. (1997) Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89, 239–250 PubMed

Sullivan W. P., Owen B. A., Toft D. O. (2002) The influence of ATP and p23 on the conformation of hsp90. J. Biol. Chem. 277, 45942–45948 PubMed

Odunuga O. O., Hornby J. A., Bies C., Zimmermann R., Pugh D. J., Blatch G. L. (2003) Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction. Molecular characterization of the critical contacts for successful binding and specificity. J. Biol. Chem. 278, 6896–6904 PubMed

Jiménez B., Ugwu F., Zhao R., Ortí L., Makhnevych T., Pineda-Lucena A., Houry W. A. (2012) Structure of minimal tetratricopeptide repeat domain protein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90. J. Biol. Chem. 287, 5698–5709 PubMed PMC

Carrigan P. E., Sikkink L. A., Smith D. F., Ramirez-Alvarado M. (2006) Domain:domain interactions within Hop, the Hsp70/Hsp90 organizing protein, are required for protein stability and structure. Protein Sci. 15, 522–532 PubMed PMC

Hernández M. P., Sullivan W. P., Toft D. O. (2002) The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex. J. Biol. Chem. 277, 38294–38304 PubMed

Chen S., Smith D. F. (1998) Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery. J. Biol. Chem. 273, 35194–35200 PubMed

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