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Mechanism of allosteric signaling in Hsp70 nucleotide binding domains
Abstract
Hsp70 molecular chaperones have key functions in the cell for folding, repair and degradation of proteins. Hsp70s are a conserved family of proteins that consist of an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain. ATP binding leads to reduced substrate-binding affinity, and reciprocally, substrate binding activates their ATPase rate. This interdomain communication is essential for Hsp70 chaperone function. Previous work suggests a key role for a stretch of conserved hydrophobic residues, 389VLLL 392, in the interdomain linker of E. coli Hsp70, DnaK, in allosteric coupling. Strikingly, we observed that the presence of these residues on the ATPase domain of DnaK (NBD392) led to a markedly enhanced rate of hydrolysis relative to either the intact DnaK protein or a construct lacking this segment (NBD388). Thermal denaturation experiments showed differences in the stability of the two ATPase domain constructs and indicated altered rigid-body movements of subdomains upon linker interactions with the ATPase domain at pH 7.0. We have further illustrated by charge state distributions with ESI-MS (electrospray ionization mass spectrometry) that the linker docking causes conformational rearrangements to a more closed conformation at this pH. We also found that this conformational switch can be reversed by varying the pH. H/D exchange mass spectroscopy of the two ATPase domain constructs revealed different exchange kinetics for the constructs in the nucleotide free-state at pD 7.6. NBD388 displayed a single slowly-exchanging population, whereas NBD392 showed two populations: one which exchanged at a rate similar to NBD388, and one which exchanged rapidly, suggestive of a global unfolding event. The differences in exchange kinetics were reversed by the addition of ADP. These data confirmed our previous findings that NBD392 exists in dynamic equilibrium between an "open", more globally dynamic state and a "closed", less dynamic state, whereas NBD388 samples only the more "open" state. Taking all of our results together, we hypothesize a possible mechanism of allostery: Ligand-induced shift in an open/closed state equilibrium along with dynamic changes in the ATPase domain may be communicated to the substrate-binding domain via the linker residues, implicating the conserved linker as an allosteric switch.
Subject Area
Biophysics|Biochemistry|Molecular biology
Recommended Citation
Dinler, Gizem, "Mechanism of allosteric signaling in Hsp70 nucleotide binding domains" (2006). Doctoral Dissertations Available from Proquest. AAI3206216.
https://scholarworks.umass.edu/dissertations/AAI3206216