Dissertations, Theses, and Capstone Projects

Date of Degree

9-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biology

Advisor

Daniel A Keedy

Committee Members

Neel Shah

Reza Khayat

Shaneen Singh

Eta Isiorho

Subject Categories

Biochemistry | Biophysics | Molecular Biology | Other Biochemistry, Biophysics, and Structural Biology | Structural Biology

Keywords

allostery, drug discovery, structural biology, computational biology, obesity and diabetes

Abstract

Protein Tyrosine Phosphatases (PTPs) are critical regulators of cellular signaling, and their activity is often modulated through allosteric mechanisms. Allostery is defined as a chemical or conformational change in one site of the protein due to a binding event at another site. Although several studies have investigated aspects of allostery in PTPs, the mechanistic foundations underlying allosteric regulation remain poorly understood. Despite a highly conserved catalytic domain across PTPs, regions with low or no conservation suggest potential "rewiring" of allosteric networks. This raises key questions about the universality versus individuality of allosteric mechanisms across the PTP family.

In this work, I use protein tyrosine phosphatase 1B (PTP1B) as a model system to expand its known conformational landscape by employing structural perturbations to obtain two high-quality X-ray crystallographic datasets. This includes a structure collected at room-temperature (RT) using serial synchrotron crystallography (SSX), and the other structure assumes a rare packing collected at cryo condition. These data revealed the impact of temperature and crystal packing on conformational heterogeneity and allosteric coupling.

To better interpret alternative conformations, I contributed to the development of the multiconformer modeling tool qFit, which allowed verify accurate modeling of 221 PTP catalytic domain structures. Further, residue-residue interaction networks were generated across 26 distinct PTPs using a newly developed computational tool, Residue Interaction Networks From Alternate conformations In RElated structures (RINFAIRE), uncovering patterns of interaction and flexibility indicative of rewired allosteric wiring. Functional studies using mutagenesis of highly connected network residues further linked structural variability to potential regulatory mechanisms.

Collectively, these findings offer new insights into the conformational and evolutionary complexity underlying allosteric regulation in PTPs. Furthermore, they advance the understanding of allosteric mechanisms within this enzyme family and provide a foundation for the future rational design of allosteric modulators.

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