Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

SignificanceTranscriptional coactivators and their partner transcription factors have been labeled as intrinsically disordered, fuzzy, and undruggable. We propose that the identification of conserved mechanisms of engagement between coactivators and their cognate activators should provide general principles for small-molecule modulator discovery. Here, we show that the structurally divergent coactivator Med25 forms short-lived and dynamic complexes with three different transcriptional activators and that conformational shifts are mediated by a flexible substructure of two dynamical helices and flanking loops. Analogous substructures are found across coactivators. Further, targeting one of the flexible structures with a small molecule modulates Med25–activator complexes. Thus, the two conclusions of the work are actionable for the discovery of small-molecule modulators of this functionally important protein class.

Chemically induced dimerizers (CIDs) have emerged as one of the most powerful tools for artificially regulating signaling pathways in cells; however, currently available CID systems lack the properties desired for use in regulating cellular therapies. Here, we report the development of human antibody-based chemically induced dimerizers (AbCIDs) from known small-molecule-protein complexes by selecting for synthetic antibodies that recognize the chemical epitope created by the bound small molecule. We demonstrate this concept by generating three antibodies that are highly selective for the BCL-xL-ABT-737 complex compared to BCL-xL alone. We show the potential of AbCIDs for application in regulating human cell therapies by using them to induce CRISPRa-mediated gene expression and to regulate CAR T-cell activation. We believe that the AbCIDs generated in this study will find application in regulating cell therapies and that the general method of AbCID development may lead to the creation of many new and orthogonal CIDs.

We report a new chemical genetic method for creating bivalent ligands of protein kinases. The kinase inhibitors that are generated with this methodology consist of two components: (1) a synthetic, small molecule that targets the ATP-binding cleft and (2) a peptidic ligand that enhances selectivity between kinases by targeting a secondary binding domain. A key feature of these bivalent inhibitors is that they are assembled on a protein scaffold with a chemoselective protein labeling technique. The utility of this methodology is demonstrated through the generation of a panel of protein-small molecule conjugates that simultaneously target the SH1 and SH3 domains of the closely related tyrosine kinases Src and Abl. The assembled bivalent ligands are significantly more potent inhibitors of Src and Abl than either modular component alone. Importantly, these protein-small molecule conjugates show a high degree of selectivity for their intended kinase target.

We report the synthesis of a soluble perylene-based small molecule for use as an n-type emissive material for organic optoelectronic device applications, and demonstrate the material in a light-emitting electrochemical cell configuration.