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In the quest for new drug candidates, a pivotal phase involves identifying compounds that selectively and robustly bind to their targets to modulate activity for therapeutic effects. This modulation can manifest as inhibition, activation, or allosteric regulation, among others. A core challenge in drug discovery is detecting ligands with high binding affinity to target proteins. Techniques range from high-throughput screening and computational simulations to advanced machine learning models. Fragment-based drug discovery (FBDD), particularly using X-ray crystallography beamlines, has become a prominent method for finding initial leads for small-molecule modulators. This involves soaking potential ligands into protein crystals, followed by X-ray diffraction data analysis to detect binding fragments. Despite technological advancements enhancing throughput, variations in crystals unrelated to ligand binding hinder analysis sensitivity. We present VAE-Assisted Ligand Discovery (VALDO), a novel approach to distinguish meaningful conformational changes from unrelated crystal heterogeneity. VALDO employs a variational autoencoder (VAE) to encode crystallographic reflections into a low-dimensional space, filtering out noise and reconstructing an apo state. This facilitates the creation of difference maps crucial for identifying ligand binding. Comparative benchmarks against methods like PanDDA and Cluster4x show VALDO's superior ability to detect and estimate the pose of bound drug fragments.

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

The recent advent of crystallographic small-molecule fragment screening presents the opportunity to obtain unprecedented numbers of ligand-bound protein crystal structures from a single high-throughput experiment, mapping ligandability across protein surfaces and identifying useful chemical footholds for structure-based drug design. However, due to the low binding affinities of most fragments, detecting bound fragments from crystallographic datasets has been a challenge. Here we report a trove of 65 new fragment hits across 59 new liganded crystal structures for PTP1B, an \"undruggable\" therapeutic target enzyme for diabetes and cancer. These structures were obtained from computational analysis of data from a large crystallographic screen, demonstrating the power of this approach to elucidate many (~50% more) \"hidden\" ligand-bound states of proteins. Our new structures include a fragment hit found in a novel binding site in PTP1B with a unique location relative to the active site, one that validates another new binding site recently identified by simulations, one that links adjacent allosteric sites, and, perhaps most strikingly, a fragment that induces long-range allosteric protein conformational responses via a previously unreported intramolecular conduit. Altogether, our research highlights the utility of computational analysis of crystallographic data, makes publicly available dozens of new ligand-bound structures of a high-value drug target, and identifies novel aspects of ligandability and allostery in PTP1B.

Allostery is central to regulation of protein function, but our mechanistic understanding remains incomplete. A deeper understanding of how redistributions of conformational states drive allostery in proteins could allow us to better grasp natural regulatory principles in cells and open new doors to therapeutic development. Conformationally dynamic human Protein Tyrosine Phosphatases (PTPs) exemplify the challenges and opportunities associated with allostery. The archetypal PTP, PTP1B, has been highly validated as a therapeutic target but no allosteric inhibitors have been approved for clinical use. This is largely because our understanding of the mechanisms underlying allostery in PTP1B remains limited, despite the discovery of putative allosteric sites in PTP1B.In the work described in this dissertation, I used high-throughput small-molecule fragment soaking and room-temperature X-ray crystallography to determine how temperature affects the occupancy, pose, and location of small-molecule fragments binding to PTP1B. These structures also indicated that temperature can modulate protein conformational responses to ligand binding, leading to new insights into allosteric networks. Building on fragment-bound structures, I worked to apply structure based drug design (SBDD) methods to design allosteric molecules that can modulate PTP1B function. In particular, I designed a new approach to bias the conformational ensemble of the targeted allosteric site using multistate docking simulations. I also worked with biotech companies who applied their technologies to a SBDD approach to design potential allosteric modulators. We uncovered both allosteric inhibitors and benign binders, and I determined the crystal structure of a designed compound bound to the targeted allosteric site in PTP1B. Lastly, I used computational and molecular modeling to map clinically relevant PTP mutations onto the structures of PTP1B and a close homolog, T-Cell Protein Tyrosine Phosphatase (TCPTP), and uncovered that the mutations likely enact their functional effects by perturbing allosteric sites and allosteric networks of the protein.Overall, this research, which marries unique techniques in experimental X-ray crystallography and computational structural biology, provides unique insights into protein-ligand interactions, particularly for allosteric sites, and provides promising new allosteric ligand footholds for a biomedically important enzyme. More broadly, this work will improve our general understanding of how transitions between multiple conformational states underlie allosteric regulation.

Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal allosteric sites to target for weight loss therapy. To do so, we selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in human cells. Focusing on the variants in and near the ordered catalytic domain, we ascribed structural mechanism to their functional effects using enzyme activity assays, room-temperature X-ray crystallography, and local hydrogen-deuterium exchange mass spectrometry (HDX-MS). By combining these complementary structural biology experiments for multiple variants, we characterize an inherent allosteric network in PTP1B that differs from previously reported allosteric inhibitor-driven mechanisms mediated by catalytic loop motions. The most functionally impactful variant sites map to highly ligandable surface sites, suggesting untapped opportunities for allosteric drug design. Overall, these studies can inform the targeted design of allosteric PTP1B inhibitors for the treatment of obesity.

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy*, Hill*, 2018). Here we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly -- but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryogenic-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function. Competing Interest Statement The authors have declared no competing interest.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments including diabetes, obesity, and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between datasets including non-isomorphism. Here we present the highest-resolution crystal structure of apo wildtype (WT) PTP1B to date, out of ∼350 total PTP1B structures in the PDB. Our structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. We interrogate the conformational differences between these chains in our apo structure, and between several recently reported high-resolution ligand-bound structures. We also examine electron density maps in a high-resolution structure of a recently reported activating double mutant, and discover unmodeled alternate conformations in the mutant structure that coincide with regions of enhanced conformational heterogeneity in our new WT structure. Our results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, our new data and analysis provide a freshly detailed view of the conformational ensemble of PTP1B, and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.