title: The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex.
abstract: 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.
authors.affiliation: From the Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology, Zluty kopec 7, 656 53 Brno, Czech Republic.
authors.forename: Filip
authors.initials: F
authors.lastname: Trcka
authors.forename: Michal
authors.initials: M
authors.lastname: Durech
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: Lenka
authors.initials: L
authors.lastname: Hernychova
authors.forename: Petr
authors.initials: P
authors.lastname: Muller
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojtesek
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C112969:HSP90AA1 protein, human; C490786:HSPA1A protein, human; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes; C108485:TOMM34 protein, human
keywords: Co-chaperone; H/D Exchange; Hsp70; Hsp90; Molecular Chaperone; Protein Assembly; Protein Folding; Protein-Protein Interactions; TPR Domain