Biology subjects

Keedy, D. A.

Publications and source records attributed to Keedy, D. A..

16 recordsLinked to original sources

bioRxiv2025

Mapping allosteric rewiring in related protein structures from collections of crystallographic multiconformer models

How do related proteins with a common fold perform diverse biological functions? Although the average structure may be similar, structural excursions from this average may differ, giving rise to allosteric rewiring that enables differential activity and regulation. However, this idea has been difficult to test in detail. Here we used the qFit algorithm to model "hidden" alternate conformations from electron density maps for an entire protein family, the Protein Tyrosine Phosphatases (PTPs), spanning 26 enzymes and 221 structures. To interrogate these multiconformer models, we developed a new algorithm, Residue Interaction Networks From Alternate conformations In RElated structures (RINFAIRE), that calculates networks of interactions between flexible residues and quantitatively compares them. We show that PTPs share a common allosteric network which rewires dynamically in response to catalytic loop motions or active-site vs. allosteric ligand binding, but also that individual PTPs have unique allosteric signatures. As experimental validation, we show that targeted mutations at residues with varying sequence conservation but high network connectivity modulate enzyme catalysis, including a surprising enhancement of activity. Overall, our work provides new tools for understanding how evolution has recycled modular macromolecular building blocks to diversify biological function. RINFAIRE is available at https://github.com/keedylab/rinfaire.

biophysics↗

bioRxiv2024

Three STEPs forward: A trio of unexpected structures of PTPN5

Protein tyrosine phosphatases (PTPs) play pivotal roles in myriad cellular processes by counteracting protein tyrosine kinases. Striatal-enriched protein tyrosine phosphatase (STEP, PTPN5) regulates synaptic function and neuronal plasticity in the brain and is a therapeutic target for several neurological disorders. Here, we present three new crystal structures of STEP, each with unexpected features. These include high-resolution conformational heterogeneity at multiple sites, and a highly coordinated citrate molecule in the active site, a previously unseen conformational change at an allosteric site, an intramolecular disulfide bond that was characterized biochemically but had never been visualized structurally, and two serendipitous covalent ligand binding events at surface-exposed cysteines that are nearly or entirely unique to STEP among human PTPs. Together, our results offer new views of the conformational landscape of STEP that may inform structure-based design of allosteric small molecules to specifically inhibit this biomedically important enzyme.

biophysics↗

bioRxiv2024

Structures of human PTP1B variants reveal allosteric sites to target for weight loss therapy

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 in vitro 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. SignificanceObesity is a growing public health concern worldwide. The human enzyme PTP1B is a validated obesity drug target, but to date, effective PTP1B inhibitors have not been developed. Allosteric drugs, which target parts of a protein distant from the active site, offer advantages for specific inhibition -- but finding promising allosteric sites that bind ligands and convey allosteric signals remains challenging. To address this knowledge gap, we used human genetic studies in thin people to identify amino acid variants that diminish PTP1B function. We then used complementary structural biology methods to show that the variants exploit distinct allosteric wiring within PTP1B that includes ligand-enriched binding sites. This work demonstrates how a unique combination of genetics and biophysics can unveil promising allosteric sites in challenging drug target proteins.

biophysics↗

bioRxiv2024

Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction

The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter protein function or cell signaling. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling. Significance StatementProtein-protein interactions are critical for cell signaling. The interaction between the phosphatase PTP1B and adaptor protein Grb2 co-localizes PTP1B with its substrates, thereby enhancing their dephosphorylation. We show that Grb2 binding also directly modulates PTP1B activity through an allosteric mechanism involving the proline-rich region of PTP1B. Our study reveals a novel mode of PTP1B regulation through a protein-protein interaction that is likely to be exploited by other cellular interactors of this important signaling enzyme.

biochemistry↗

bioRxiv2024

An expanded view of ligandability in the allosteric enzyme PTP1B from computational reanalysis of large-scale crystallographic data

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.

biophysics↗

bioRxiv2023

High-resolution double vision of the archetypal protein tyrosine phosphatase

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.

biophysics↗

bioRxiv2023

Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and buriedness. We demonstrate that our HDX rate data obtained in solution adds value to predictions of dynamics derived from a pseudo-ensemble constructed from [~]200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site vs. allosteric small-molecule inhibitors. These maps reveal distinct, dramatic, and widespread effects on protein dynamics relative to the apo form, including changes to dynamics in locations distal (>35 [A]) from the respective ligand binding sites. These results help shed light on the allosteric nature of PTP1B and the surprisingly far-reaching consequences of inhibitor binding in this important protein. Overall, our work showcases the potential of HDX-MS for elucidating protein conformational dynamics and allosteric effects of small-molecule ligands, and highlights the potential of integrating HDX-MS alongside other complementary methods to guide the development of new therapeutics.

biophysics↗

bioRxiv2023

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

biophysics↗

bioRxiv2023

Pushed to extremes: distinct effects of high temperature vs. pressure on the structure of an atypical phosphatase

Protein function hinges on small shifts of three-dimensional structure. Elevating temperature or pressure may provide experimentally accessible insights into such shifts, but the effects of these distinct perturbations on protein structures have not been compared in atomic detail. To quantitatively explore these two axes, we report the first pair of structures at physiological temperature vs. high pressure for the same protein, STEP (PTPN5). We show that these perturbations have distinct and surprising effects on protein volume, patterns of ordered solvent, and local backbone and side-chain conformations. This includes novel interactions between key catalytic loops only at physiological temperature, and a distinct conformational ensemble for another active-site loop only at high pressure. Strikingly, in torsional space, physiological temperature shifts STEP toward previously reported active-like states, while high pressure shifts it toward a previously uncharted region. Together, our work argues that temperature and pressure are complementary, powerful, fundamental macromolecular perturbations.

biophysics↗

bioRxiv2022

Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

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.

biophysics↗

bioRxiv2022

Room-temperature serial synchrotron crystallography of apo PTP1B

Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but a common hurdle is obtaining sufficiently large protein crystals. Serial synchrotron crystallography (SSX) helps address this hurdle by allowing the use of many medium- to small-sized crystals. We have used a recently introduced serial sample support chip system to obtain the first SSX structure of a human phosphatase, specifically Protein Tyrosine Phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations for the active-site WPD loop and for a distal allosteric site. By contrast, in our SSX structure, the active site is best fit with a single conformation, but the distal allosteric site is best fit with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.

biophysics↗

bioRxiv2021

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics. SynopsisX-ray crystallography at variable temperature for SARS-CoV-2 Mpro reveals a complex conformational landscape, including mobile solvent at the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and an intramolecular network bridging the active site and dimer interface.

biophysics↗

bioRxiv2020

qFit 3: Protein and ligand multiconformer modeling for X-ray crystallographic and single-particle cryo-EM density maps

New X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open-source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0.

biophysics↗

bioRxiv2018

Model selection for biological crystallography

Structural biologists have fit increasingly complex model types to protein X-ray crystallographic data, motivated by higher-resolving crystals, greater computational power, and a growing appreciation for protein dynamics. Once fit, a more complex model will generally fit the experimental data better, but it also provides greater capacity to overfit to experimental noise. While refinement progress is normally monitored for a given model type with a fixed number of parameters, comparatively little attention has been paid to the selection among distinct model types where the number of parameters can vary. Using metrics derived in the statistical field of model comparison, we develop a framework for statistically rigorous inference of model complexity. From analysis of simulated data, we find that the resulting information criteria are less likely to prefer an erroneously complex model type and are less sensitive to noise, compared to the crystallographic cross-validation criterion Rfree. Moreover, these information criteria suggest caution in using complex model types and for inferring protein conformational heterogeneity from experimental scattering data.

biophysics↗

bioRxiv2018

qFit-ligand reveals widespread conformational heterogeneity of drug-like molecules in X-ray electron density maps

Proteins and ligands sample a conformational ensemble that governs molecular recognition, activity, and dissociation. In structure-based drug design, access to this conformational ensemble is critical to understand the balance between entropy and enthalpy in lead optimization. However, ligand conformational heterogeneity is currently severely underreported in crystal structures in the Protein Data Bank, owing in part to a lack of automated and unbiased procedures to model an ensemble of protein-ligand states into X-ray data. Here, we designed a computational method, qFit-ligand, to automatically resolve conformationally averaged ligand heterogeneity in crystal structures, and applied it to a large set of protein receptor-ligand complexes. We found that up to 29 % of a dataset of protein crystal structures bound with drug-like molecules present evidence of unmodeled, averaged, relatively isoenergetic conformations in ligand-receptor interactions. In many retrospective cases, these alternate conformations were adventitiously exploited to guide compound design, resulting in improved potency or selectivity. Combining qFit-ligand with high-throughput screening or multi-temperature crystallography could therefore augment the structure-based drug design toolbox.

biophysics↗

bioRxiv2017

New routes for PTP1B allosteric inhibition by multitemperature crystallography, fragment screening and covalent tethering

Allostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new 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 a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.

biophysics↗