Using Steered Molecular Dynamics to Predict and Assess Hsp70 Substrate-Binding Domain Mutants that Alter Prion Propagation

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. Author Summary: Direct non-covalent interactions between protein substrates and molecular chaperones play crucial roles in the protein folding process. [PSI+] is a prion of the yeast Saccharomyces cerevisiae, which is formed by mis-folding of the native Sup35 protein in a process analogous to formation of prions in mammals. While much genetic data exists showing a clear role for Hsp70 in prion propagation, biochemical data has yet to provide a clear link to how Hsp70 functions in prion propagation or how some Hsp70 mutants successfully impair in vivo propagation of prions. This paper employs a novel simulation method termed “steered molecular dynamics” to explore the different types and amounts of non-covalent interactions between wild type and mutated Hsp70, with a model substrate. Extrapolating the in silico data allowed us to decipher how a mutant Hsp70 impairs yeast prion propagation and allows us to predict other Hsp70 mutants that should behave in the same manner and to test these predictions in a yeast-based system. Our computational data shows that increasing the binding affinity of Hsp70 for substrate is one way of impairing prion propagation, a proposal that correlates very well with previous experimental genetic data.