title: Identification of key hinge residues important for nucleotide-dependent allostery in E. coli Hsp70/DnaK.
abstract: DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK's nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these "hinge" residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Peter Man-Un
authors.initials: PM
authors.lastname: Ung
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Thompson
authors.forename: Lyra
authors.initials: L
authors.lastname: Chang
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.forename: Heather A
authors.initials: HA
authors.lastname: Carlson
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009711:Nucleotides; C063842:dnaK protein, E coli