Protein tyrosine phosphatases (PTPs) are an important class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevant-and, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase 1B (PTP1B) with bioinformatic analyses of the PTP family to examine allosteric communication in PTPs. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP1B possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from kinetic studies and mutational analyses show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations. File list (2) download file view on ChemRxiv Manuscript Revised.pdf (12.05 MiB) download file view on ChemRxiv Supporting Information Revised.pdf (17.02 MiB)

Supplementary Appendix 1. Analysis of the crystal structure of PTP1BPS. We used X-ray crystallography to examine the structural integrity of PTP1B within the PTP1BPS chimera (Supplementary Table 3). Crystals of PTP1BPS exhibited three surprising features ( Fig. 1d and Supplementary Fig. 2): (i) Their unit cell and space group were indistinguishable from those of previously collected crystals of PTP1B and PTP1B-ligand complexes. (ii) They permitted resolution of PTP1B, but not LOV2. (iii) They showed PTP1B with a wild-type conformation (i.e., the root-mean-square deviation of aligned atoms between the catalytic domains of PTP1BPS and wild-type PTP1B was 0.30 Å). These features, considered alone, might indicate that LOV2 is absent from our crystals; four additional crystallographic attributes, however, contradict this interpretation: (i) Our crystals were yellow, a color derived from LOV2 (a consequence of its FMN cofactor 1,2 ; Supplementary Figures 2a and 2b). (ii) When exposed to 455-nm light, the crystals turned clear, an indication that LOV2 remains capable of forming a cysteine adduct with FMN ( Supplementary Figures 2d and 2e). We note: Solutions of PTP1BPS(C450M), which cannot form the cysteine adduct, do not photoswitch ( Supplementary Figure 2f). (iii) The unit cell had a gap near the ⍺7 helix, the attachment point of LOV2 ( Supplementary Figure 2c). (iv)Previously examined apo structures of PTP1B in which the ⍺7 helix is disordered possess the same unit cell and space group as our crystals. 3 These features, taken together, suggest that disorder in the ⍺7 helix of PTP1B causes variability in the orientation of LOV2 within the crystal lattice. Broadly, PTP1B crystallizes with the same space group, unit cell, and conformation in the presence and absence of LOV2 and, thus, appears to be structurally unperturbed (to the extent detectable with X-ray crystallography) by this light-sensitive fusion partner.

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge-the inhibition of a human drug target-into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identi ed two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors target an allosteric site, which confers unusual selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-speci c detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely de ned biochemical activities yet possess unanticipated structures and/or binding sites.

The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge—the inhibition of a human drug target—into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.