title: The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change.
abstract: The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.
authors.affiliation: Beckman Laboratories for Structural Biology, Department of Structural Biology, Stanford University School of Medicine, California 94305-5400, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Sousa
authors.forename: D B
authors.initials: DB
authors.lastname: McKay
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D014965:X-Rays
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D012996:Solutions; D013912:Threonine; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases