Aligned variants were observed in any of three biological replicates. (A) Alignment of PA variants to secondary structure, relative surface accessibility, and site entropy. Residues 560–660 are shown, highlighting a mutational hotspot that occurs in all proteostasis environments outlined in red. (B) Alignment of PB1 variants to secondary structure, relative surface accessibility, and site entropy. Residues 1–100 are shown, highlighting a mutational hotspot observed only when HSF1 is activated outlined in purple. (C) Mutational hotspots mapped onto the PA-PB1 complex crystal structure (PDBID 4WSB) (Reich et al., 2014). PA hotspot is shaded red; PB1 hotspot is shaded purple. (D) List of amino acid substitutions likely to affect PA-PB1 binding that appear in the HSF1-activated and/or Hsp90-inhibited environments. (E) Binding free energy* of the PAC–PB1N complex (kcal/mol; calculated as the contribution (PAC–PB1N complex) – contribution (PAC) – contribution (PB1N)). Binding free energies shown for simulations with wild-type and mutant subunits; reported error is SEM. *Excludes contribution from solute configuration entropy. Figure 5—source data 1. Energy contributions from molecular dynamics simulations.