Quaternary structure of the HSC70 cochaperone HIP

HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone inv olved in the regulation of HSC70 chaperone activity and the maturation of p rogesterone receptor. To determine the quaternary structure and the gross c onformation of the protein in solution, a wide array of biochemical and bio physical techniques has been used. Size-exclusion chromatography and sedime ntation velocity indicate the presence of a single species with a Stokes ra dius, R-s, of 55 Angstrom and a sedimentation coefficient, S degrees(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 concentra tions and rotor speeds, gives a molecular mass of 88 284 Da, almost in exac t agreement with the molecular mass of a dimer. On the basis of these data, a frictional ratio f/f(o) of 1.6 is obtained, suggesting an elongated shap e for the HIP dimer. Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all alpha-protein, a ble to form extended coiled coils. Using threading and comparative model bu ilding methods, a structural model of a segment of HIP involved in HSC70 bi nding has been constructed and potential sites of interaction between HIP a nd HSC70 are proposed on the basis of electrostatic as well as shape comple mentarity. Altogether, these results indicate that HIP is an elongated dime r, able to bind two HSC70 molecules through its TPR regions, and suggest th e existence of a versatile binding site on HSC70 that may be involved in th e interaction of the chaperone with the cochaperones or other interacting p roteins.