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TRAF6 is an adapter protein and E3 ubiquitin ligase involved in signaling downstream of cell receptors essential for development and the immune system. TRAF6 participates in many protein-protein interactions, some of which are mediated by a C-terminal MATH domain that recruits TRAF6 to cell-surface receptors and associated proteins. The TRAF6 MATH domain binds to short peptide segments containing the motif PxExx[FYWHDE], where x is any amino acid. Blocking TRAF6 interactions is associated with favorable effects in various disease models. To better define the TRAF6 MATH domain binding preferences, we generated a bacterial cell-surface peptide display library to sample the TRAF6 motif sequence space. We performed sorting experiments and identified 236 of the best TRAF6-interacting peptides and a set of 1,200 peptides that match the sequence PxE but do not bind TRAF6. Selected binders, tested by single-clone bacterial display titrations and bio-layer interferometry, bound TRAF6 tighter than previously measured native peptides. To elucidate the structural basis for TRAF6 interaction preferences, we built all-atom structural models of the TRAF6 MATH domain in complex with high-affinity binders and motif-matching nonbinders that were identified in the screen. We identified motif features that favor binding to TRAF6 as well as negative design elements distributed across the motif that can disfavor or preclude binding. Searching the human proteome for matches to the library screening-defined binding motif revealed that most known, biologically relevant TRAF6 motif matches occupy a different sequence space from the most enriched hits discovered in combinatorial library screening. Our experimentally determined binding preferences and structural models can support the design of peptide-based interaction inhibitors with higher affinities than endogenous TRAF6 ligands.

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.

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.

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 fluorescence resonance energy transfer experiments measuring arm closure rates of Hsp90. Our observations suggest that ADP at physiological levels is important to Hsp90 structure, activity, and regulation.

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 reticulum-specific 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.