Quaternary Structure of the HSC70 Cochaperone HIP

• Published in: Biochemistry (Easton) Vol. 39; no. 2; pp. 307 - 315
• Main Authors: Velten, Marion, Villoutreix, Bruno O, Ladjimi, Moncef M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18-01-2000
• Subjects: Amino Acid Sequence; Binding Sites; Carrier Proteins - chemistry; Centrifugation, Density Gradient; Chromatography, Gel; Dimerization; Escherichia coli - metabolism; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones - biosynthesis; Molecular Chaperones - chemistry; Molecular Sequence Data; Molecular Weight; Protein Conformation; Protein Structure, Quaternary; Sequence Alignment
• DOI: 10.1021/bi9917535
• EISSN: 1520-4995
• ISSN: 0006-2960

HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor. To determine the quaternary structure and the gross conformation of the protein in solution, a wide array of biochemical and biophysical techniques has been used. Size-exclusion chromatography and sedimentation velocity indicate the presence of a single species with a Stokes radius, R s, of 55 Å and a sedimentation coefficient, s°20,w, of 4.34 S. The combination of these data gives a molecular mass of 101 000 Da, a value close to that of the theoretical molecular mass of a dimer (87 090 Da). Sedimentation equilibrium, performed at various protein concentrations and rotor speeds, gives a molecular mass of 88 284 Da, almost in exact agreement with the molecular mass of a dimer. On the basis of these data, a frictional ratio f/f 0 of 1.6 is obtained, suggesting an elongated shape for the HIP dimer. Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all α-protein, able to form extended coiled coils. Using threading and comparative model building methods, a structural model of a segment of HIP involved in HSC70 binding has been constructed and potential sites of interaction between HIP and HSC70 are proposed on the basis of electrostatic as well as shape complementarity. Altogether, these results indicate that HIP is an elongated dimer, able to bind two HSC70 molecules through its TPR regions, and suggest the existence of a versatile binding site on HSC70 that may be involved in the interaction of the chaperone with the cochaperones or other interacting proteins.