Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

• Published in: Archives of biochemistry and biophysics 2011-09, Vol.513 (2), p.119-125
• Main Authors: Gava, Lisandra M., Gonçalves, Danieli C., Borges, Júlio C., Ramos, Carlos H.I.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical ultracentrifugation; Biophysical Phenomena; cytosol; Dimerization; heat shock proteins; Hsp90; HSP90 Heat-Shock Proteins - chemistry; HSP90 Heat-Shock Proteins - genetics; HSP90 Heat-Shock Proteins - metabolism; Humans; In Vitro Techniques; Isothermal titration calorimetry; Kinetics; mitochondria; Mitochondrial Membrane Transport Proteins - chemistry; Mitochondrial Membrane Transport Proteins - genetics; Mitochondrial Membrane Transport Proteins - metabolism; Models, Molecular; nuclear genome; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Folding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein translocation into mitochondria; protein transport; Protein–protein interaction; receptors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; stoichiometry; Thermodynamics; Tom70; translation (genetics)
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2011.06.015

SEC–MALS combined with SDS–PAGE for the determination of the molecular mass of the Tom70 C-Hsp90 complex. Free C-Hps90 presented a MM consistent with a dimer, free Tom70 presented a MM consistent with a monomer and mixed Tom70 C-Hsp90 presented a MM indicating a stoichiometry of one monomer of Tom70 to a dimer of C-Hsp90 in the complex. [Display omitted] ► The interaction of human proteins C-Hsp90 and Tom70 was studied by biophysical tools. ► One monomer of Tom70 binds a dimer of C-Hsp90 with a KD of 360±30nM. ► Tom70 has a high affinity for C-Hsp90 in comparison to other TPR proteins. A large majority of the 1000–1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a KD of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.