Macromolecular function is rooted in energy landscapes, where sequence determines not a single structure but an ensemble of conformations. Hence, evolution modifies a protein’s function by altering its energy landscape. Here, we recreate the evolutionary pathway between two modern human oncogenes, Src and Abl, by reconstructing their common ancestors. Our evolutionary reconstruction combined with x-ray structures of the common ancestor and pre–steady-state kinetics reveals a detailed atomistic mechanism for selectivity of the successful cancer drug Gleevec. Gleevec affinity is gained during the evolutionary trajectory toward Abl and lost toward Src, primarily by shifting an induced-fit equilibrium that is also disrupted in the clinical T315I resistance mutation. This work reveals the mechanism of Gleevec specificity while offering insights into how energy landscapes evolve.

Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulumspecific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro.Molecular chaperones play a central role in maintaining folded and active proteins in the cell. Hsp70 class chaperones inhibit misfolding by binding and releasing short hydrophobic segments of unstructured polypeptides with cycles of ATP hydrolysis. Hsp60 class chaperonins promote folding by isolating single protein chains within an isolated cavity. Similar to Hsp70 and Hsp60, Hsp90 has an essential ATPase activity, but the underlying functional mechanism appears to be different. Hsp90 plays important regulatory roles under non-stress conditions by its interactions with specific classes of substrates ("clients") such as kinases and nuclear receptors (1). Hsp90 has a dimeric structure that can undergo dramatic rearrangements upon ATP binding and hydrolysis. Despite significant progress in characterizing Hsp90's ATP-dependent conformational cycle in vitro, it is still unclear how Hsp90 performs its many critical cellular functions.Hsp90 conformational heterogeneity results from rigid-body rearrangements of the three domains within the monomer. The N-terminal domain (site of nucleotide binding) can rotate relative to the middle domain. The middle domain can be positioned against the C-terminal domain (site of dimerization) in multiple orientations, resulting in a flexible and structurally heterogeneous open conformation. Indeed, all full-length Hsp90 str...

The ATPase cycle of the Hsp90 molecular chaperone is essential for maintaining the stability of numerous client proteins. Extensive analysis has focused on ATP-driven conformational changes of Hsp90, however, little is known about how Hsp90 operates under physiological nucleotide conditions in which both ATP and ADP are present. By quantifying Hsp90 activity under mixed nucleotide conditions we find dramatic differences in ADP-sensitivity among Hsp90 homologs. ADP acts as a strong ATPase inhibitor of cytosol-specific Hsp90 homologs, whereas organellular Hsp90 homologs (Grp94 and TRAP1) are relatively insensitive to the presence of ADP. These results imply that an ATP/ADP heterodimer of cytosolic Hsp90 is the predominant active state under physiological nucleotide conditions. ADP-inhibition of human and yeast cytosolic Hsp90 can be relieved by the cochaperone aha1. ADP-inhibition of bacterial Hsp90 can be relieved by bacterial Hsp70 and an activating client protein. These results suggest that altering ADP-inhibition may be a mechanism of Hsp90 regulation. To determine the molecular origin of ADP-inhibition, we identify residues that preferentially stabilize either ATP or ADP. Mutations at these sites can both increase and decrease ADP-inhibition. An accounting of ADP is critically important for designing and interpreting experiments with Hsp90. For example, contaminating ADP is a confounding factor in FRET experiments measuring arm closure rates of Hsp90. Our observations suggest that ADP at physiological levels is important to Hsp90 structure, activity, and regulation.

While cytosolic Hsp70 and Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp70 and Hsp90 paralogs (BiP and Grp94) recognize clients and influence their folding. Here, we examine how BiP and Grp94 influence the folding of insulin-like growth factor 2 (IGF2). Full-length proIGF2 is composed of an insulin-like hormone and an E-peptide that has sequence characteristics of an intrinsically disordered region. We find that the E-peptide region allows proIGF2 to form oligomers. BiP and Grp94 influence both the folding and the oligomerization of proIGF2. BiP and Grp94 exert a similar holdase function on proIGF2 folding by preferentially binding the proIGF2 unfolded state, rather than stabilizing specific folding intermediates and changing the proIGF2 folding process. In contrast, BiP and Grp94 exert counteracting effects on proIGF2 oligomerization. BiP suppresses proIGF2 oligomerization under both ADP and ATP conditions. Interestingly, Grp94 can enhance proIGF2 oligomerization whenGrp94 adopts an open conformation (ADP conditions), but not when Grp94 is in the closed conformation (ATP conditions). We propose that BiP and Grp94 regulate the assembly of proIGF2 oligomers, and that regulated oligomerization may enable proIGF2 to be effectively packaged for export from the ER to the Golgi.