SOLUTION SMALL-ANGLE X-RAY-SCATTERING STUDY OF THE MOLECULAR CHAPERONE HSC70 AND ITS SUBFRAGMENTS

Solution X-ray scattering experiments have been carried out on recombi nant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase- and peptide-binding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptide-binding fragment (residues 388-554). Modeling based on stea dy-state values of radii of gyration (R(g)'s) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments whil e Hsc70 and the 60 kDa fragment are prolate and relatively elongated. R(g) values decrease significantly in the presence of MgATP relative t o their values in the presence of MgADP (Delta R(g) similar to 4-5 Ang strom) for Hsc70 and the 60 kDa fragment; in contrast, they are essent ially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of R(g) for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transitio n to the ATP-induced R(g) occurs significantly more rapidly than ATP h ydrolysis while the reverse transition to the larger R(g) value does n ot occur before product release. Altogether, the solution scattering d ata support a model in which a conformational change in Hsc70 (presuma bly to the low-peptide-affinity state) is predicated on ATP binding wh ile the reverse transition is predicated on product release.