Dissertations, Theses, and Capstone Projects

Date of Degree

2-2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Daniel Keedy

Committee Members

Rinat Abzalimov

Thomas Kurtzman

Arvin Dar

Jean Gaffney

Subject Categories

Biochemistry | Biophysics | Enzymes and Coenzymes | Medicinal and Pharmaceutical Chemistry | Organic Chemicals | Pharmaceutics and Drug Design | Structural Biology

Keywords

Allostery, hydrogen-deuterium exchange mass spectrometry, room-temperature crystallography, PTP1B, protein tyrosine phosphatase, drug discovery

Abstract

Allostery is a pervasive regulatory principle throughout biology, yet the structural pathways by which distal inputs alter active‑site chemistry within protein structures remain incompletely defined and exploited. This dissertation uses protein tyrosine phosphatase 1B (PTP1B) as a tractable model to map those pathways with complementary experimental and computational tools. I integrate high‑resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), room-temperature crystallography, NMR, steady-state kinetics, crystallographic pseudo-ensembles, and machine-learning-guided ligand discovery. The working premise is that regulation reflects redistribution within a conformational ensemble rather than a binary switch. That orthogonal perturbations by small molecules, mutations, and protein partners can be used to both delineate and control the underlying network. HDX‑MS establishes a solution‑state baseline for PTP1B dynamics and quantifies long‑range responses to ligands, while pseudo‑ensembles provide structural context for interpreting those dynamics. Together, these methods create a host of sensitive readouts for coupling among catalytic loops and remote surfaces.

Applying this framework, I first compare an active‑site inhibitor (TCS401) with an established allosteric ligand (BB3). Both depress exchange in the Q and WPD loops, with BB3 eliciting broader, distal changes consistent with entropic compensation, thereby extending the known allosteric network beyond prior assignments. I then use biophysical and structural techniques to probe four rare PTPN1 gene variants identified from an outlying lean cohort and the UK Biobank, altering PTP1B function in cells. These biophysical experiments trace the most potent effects to remote structural determinants that perturb catalytic-loop behavior. Several high‑impact positions cluster on surface pockets that prove ligandable, nominating previously untapped regulatory footholds. In a physiological counterpoint, the adaptor Grb2 binds the PTP1B proline‑rich region (PRR) and directly increases kcat across multiple phosphopeptide substrates without substrate colocalization. NMR and proteomics support specific PRR-dependent engagement and stabilization of catalytic-domain elements, demonstrating scaffold-mediated allostery in a phosphatase that lacks a dedicated regulatory domain. Finally, I evaluate a distal pocket centered on Loop‑16 (L16). The P241G mutation increases kcat by approximately 25% with no change in Km, consistent with V‑type modulation transmitted from L16 to the active site. A multiconformer AtomNet screen yields eleven reproducible binders, dominated by an imidazo‑pyridine series; most are functionally silent, but Atw103 inhibits noncompetitively with an HDX signature marked by E‑, pTyr‑recognition, and Q‑loop protection, consistent with distal transduction. A thermally derived species, Atw47B, shows an active‑site‑centered HDX pattern and lot dependence, cautioning against misassignment.

These results support a model of PTP1B as a distributed regulatory system with multiple ligandable surfaces that can be read and written through ensemble redistribution, rather than simpler models of conformational switching. Conceptually, it concludes with thematic motivations that align with my future research interests in multivalency, aiming to convert weak recognition of individual small molecule ligands into precise situational control of disease targets. I outline translational strategies by others that include bidentate ligands bridging adjacent pockets to encode cooperativity and selectivity, and event‑driven proximity modalities such as PROTACs and PhosTACs to extend control across proteins. I also propose explicit tests for combinatorial allostery at multiple PTP1B sites using full concentration-response surfaces fit to two-site models with cooperativity terms, enabling logic-like integration when pockets are co-occupied. These principles generalize to other dynamic enzymes and provide a framework for selective pharmacology, especially in phosphatases.

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