title: Using steered molecular dynamics to predict and assess Hsp70 substrate-binding domain mutants that alter prion propagation.
abstract: Genetic screens using Saccharomyces cerevisiae have identified an array of cytosolic Hsp70 mutants that are impaired in the ability to propagate the yeast [PSI(+)] prion. The best characterized of these mutants is the Ssa1 L483W mutant (so-called SSA1-21), which is located in the substrate-binding domain of the protein. However, biochemical analysis of some of these Hsp70 mutants has so far failed to provide major insight into the specific functional changes in Hsp70 that cause prion impairment. In order to gain a better understanding of the mechanism of Hsp70 impairment of prions we have taken an in silico approach and focused on the Escherichia coli Hsp70 ortholog DnaK. Using steered molecular dynamics simulations (SMD) we demonstrate that DnaK variant L484W (analogous to SSA1-21) is predicted to bind substrate more avidly than wild-type DnaK due to an increase in numbers of hydrogen bonds and hydrophobic interactions between chaperone and peptide. Additionally the presence of the larger tryptophan side chain is predicted to cause a conformational change in the peptide-binding domain that physically impairs substrate dissociation. The DnaK L484W variant in combination with some SSA1-21 phenotypic second-site suppressor mutations exhibits chaperone-substrate interactions that are similar to wild-type protein and this provides a rationale for the phenotypic suppression that is observed. Our computational analysis fits well with previous yeast genetics studies regarding the functionality of the Ssa1-21 protein and provides further evidence suggesting that manipulation of the Hsp70 ATPase cycle to favor the ADP/substrate-bound form impairs prion propagation. Furthermore, we demonstrate how SMD can be used as a computational tool for predicting Hsp70 peptide-binding domain mutants that impair prion propagation.
authors: Province Key Laboratory of Animal Resource and Epidemic Disease Prevention, Liaoning University, Shenyang, China.
authors: Linan
authors: L
authors: Xu
authors: Naushaba
authors: N
authors: Hasin
authors: Manli
authors: M
authors: Shen
authors: Jianwei
authors: J
authors: He
authors: Youlin
authors: Y
authors: Xue
authors: Xiaohong
authors: X
authors: Zhou
authors: Sarah
authors: S
authors: Perrett
authors: Youtao
authors: Y
authors: Song
authors: Gary W
authors: GW
authors: Jones
mesh_terms: D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011328:Prions / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D018840:HSP70 Heat-Shock Proteins; D011328:Prions
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