Physics-based modeling provides predictive understanding of selectively promiscuous substrate binding by Hsp70 chaperones

Author summary Molecular chaperones are proteins that help prevent misfolding and aggregation of their substrates. This is a complex task, as the cell is very crowded, and the chaperone must efficiently bind to misfolded regions of the client protein while avoiding well-folded proteins. An additional confounding detail is that proteins can enter the binding cleft of some Hsp70s in two orientations, a fact often unaccounted for by existing sequence-based models. Here, we developed a model to describe how client proteins bind to DnaK, the E. coli Hsp70, using physics-based molecular dynamics simulations to quantify the interactions between a variety of peptide substrates and key sites on DnaK. The resulting model, which we call Paladin, provides a physical basis to understand how DnaK binds to specific peptides. Given a sequence, Paladin can predict the precise residues that bind at a specific site on DnaK and can further explain challenging features like the binding orientation, which are typically not predicted by sequence-only models. The Paladin model could be used to design 'super-binder' therapeutic peptides to inhibit chaperones like DnaK in E. coli. To help cells cope with protein misfolding and aggregation, Hsp70 molecular chaperones selectively bind a variety of sequences ("selective promiscuity"). Statistical analyses from substrate-derived peptide arrays reveal that DnaK, the E. coli Hsp70, binds to sequences containing three to five branched hydrophobic residues, although otherwise the specific amino acids can vary considerably. Several high-resolution structures of the substrate -binding domain (SBD) of DnaK bound to peptides reveal a highly conserved configuration of the bound substrate and further suggest that the substrate-binding cleft consists of five largely independent sites for interaction with five consecutive substrate residues. Importantly, both substrate backbone orientations (N- to C- and C- to N-) allow essentially the same backbone hydrogen-bonding and side-chain interactions with the chaperone. In order to rationalize these observations, we performed atomistic molecular dynamics simulations to sample the interactions of all 20 amino acid side chains in each of the five sites of the chaperone in the context of the conserved substrate backbone configurations. The resulting interaction energetics provide the basis set for deriving a predictive model that we call Paladin (Physics-based model of DnaK-Substrate Binding). Trained using