Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction: molecular characterization of the critical contacts for successful binding and specificity.

Odunuga, O.O. and Hornby, J.A. and Bies, C. and Zimmerman, R. and Pugh, D.J. and Blatch, G.L. (2003) Tetratricopeptide repeat motif-mediated Hsc70-mSTI1 interaction: molecular characterization of the critical contacts for successful binding and specificity. Journal of Biological Chemistry, 278 (9). pp. 6896-6904. ISSN 0021-9258

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Official URL: http://dx.doi.org/10.1074/jbc.M206867200

Abstract

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.

Item Type:Article
Uncontrolled Keywords:Hydrophobicity; Mutagenesis; Precipitation (chemical); Proteins; Spectroscopic analysis; Substitution reactions; Stress inducible proteins; 3T3 Cells; Amino Acid Motifs; Amino Acid Sequence; Animals; Asparagine; Circular Dichroism; Fibroblasts; Gene Deletion; Glutathione; Glutathione Transferase; Heat-Shock Proteins; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Kinetics; Lysine; Mice; Models, Molecular; Molecular Chaperones; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oligonucleotides; Peptides; Precipitin Tests; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sepharose; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Surface Plasmon Resonance; Time Factors
Subjects:Y Unknown > Subjects to be assigned
Divisions:Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology
ID Code:1402
Deposited By: Mrs Eileen Shepherd
Deposited On:12 Jun 2009
Last Modified:06 Jan 2012 16:20
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