Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.

We examine the folding-unfolding of a variant of the Trp-cage, known as TC10b, and compare structural stability, dynamics, and thermodynamics with that of the TC5b variant, using replica exchange molecular dynamics (REMD). The TC10b variant was designed to have larger helical stability by the substitution of amino acids with greater alpha helical propensities in the N-terminal region. Experiments have shown TC10b to possess larger overall stability than TC5b. Simulations starting from unbiased, unfolded initial conditions are run for 1 μs per replica. The calculations show a higher melting temperature for TC10b than TC5b, and suggest a more ordered folded structure through the elimination of a substate found in the folded ensemble of TC5b. We model the difference in Gibbs free energy, ΔG(P,T), of folding using the bootstrap statistical method, which is used to calculate uncertainties associated with the thermodynamic parameters for both variants of the Trp-cage. We find that while the shape of the area for which the protein is stability folded is elliptical for TC5b, there is a degree of uncertainty associated with that of TC10b, with one model suggesting elliptical and another suggesting hyperbolic. This model suggests that at high pressures, TC5b can experience pressure denaturation, but TC10b may not.

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 available peptide array data, Paladin can distinguish binders and nonbinders of DnaK with accuracy comparable to existing predictors and further predicts the detailed configuration of the bound sequence. Tested using existing DnaK-peptide structures, Paladin correctly predicted the binding register in 10 out of 13 substrate sequences that bind in the N- to C- orientation, and the binding orientation in 16 out of 22 sequences. The physical basis of the Paladin model provides insight into the origins of how Hsp70s bind substrates with a balance of selectivity and promiscuity. The approach described here can be extended to other Hsp70s where extensive peptide array data is not available.

We study the energy landscape and thermodynamics of the zwitterionic variant of the widely studied TC5b Trp-cage protein using replica exchange molecular dynamics simulations. We show that the addition of two charge groups at the termini has dramatic consequences to the folding landscape. First, the addition of charged ends increases the equilibration time of the simulation by a factor of 2.5 over a variant with terminal capping. Second, we identify the formation of two long-lived metastable states not present in the capped ends variant structural ensemble. The population of these metastable states is higher at lower temperatures; furthermore, these states are determined to be low energy states, relative to the folded state. The first of the metastable states is a folding intermediate structure which is characterized by a non-native charge pair. The second is characterized by significant β sheet content. We show through potential of mean force (PMF) calculations that the PMF between two charge groups is a poor predictor of the prevalence of a particular ion pair in the unfolded structural ensemble. Finally, by analyzing the energy differences between the folded state, unfolded states, and the metastable states, we show that the stabilization of these metastable states is not only due to favorable Coulomb interactions but also due to strain in the dihedral angles. Our results show that, even for a simple protein, the folding landscape can be extremely complex and significantly altered by simple changes to the charge states of the sequence.