Date of Award

Spring 2022

Project Type

Dissertation

Program or Major

Molecular and Evolutionary Systems Biology

Degree Name

Doctor of Philosophy

First Advisor

Rick Cote

Second Advisor

Krisztina Varga

Third Advisor

David Plachetzki

Abstract

ABSTRACT

Structural and Biochemical Characterization of the Allosteric Regulation of Photoreceptor Phosphodiesteraseby Richa Garg

The photoreceptor phosphodiesterase (PDE6) is the central enzyme in the visual signaling pathway. Cone PDE6 is a homodimer consisting of a catalytic dimer (Pα’α’) and two cone-specific inhibitory γ-subunits (Pγ’). Rod PDE6 is a heterotetramer which consists of a catalytic dimer (Pαβ) with two rod-specific Pγ subunits. The PDE6 differs from other phosphodiesterases (PDEs), in its unique inhibitory Pγ subunit and its activation by α-subunit of the G-protein (Gα). Regulation of PDE6 activity is a key factor in determining the electric response upon light stimulations. PDE6 activity is inhibited by its Pγ subunit, activated by Gα and allosterically regulated by its regulatory domains. The central hypothesis of my research is that binding of Pγ and cGMP to PDE6 induces conformational changes in its regulatory domain that contribute to PDE6 regulation. The specific aims of my thesis are: (1) define the allosteric communication pathway of cone PDE6 upon ligand binding of cGMP and Pγ and, (2) identify the function of the N-terminal region of cone PDE6. The first aim of my research is to understand the allosteric regulation of PDE6 mediated through two tandem GAF domains upon binding of cGMP and Pγ. To address this goal, I cloned and purified chicken cone GAFab 42-458 regulatory domain (lacking first 41 N-terminal residues) and truncated cone Pγ 1-58. In collaboration with Dr. Hengming Ke (University of North Carolina), the chicken cone GAFab 42-458 was crystallized, and the X-ray structure was solved at 3.3 Å resolution. Using chemical cross-linking and mass spectrometry (CL-MS) analysis in conjunction with the Integrative Modeling Platform (IMP), I generated structural model of Apo-GAFab, cGMP bound GAFab and GAFab bound to Pγ in the presence and absence of cGMP. Comparison of these models revealed conformational changes induced within the GAFa and GAFb domains upon ligand binding of Pγ and/or cGMP. In addition, I also performed solution NMR spectroscopy (in collaboration with Dr. Krisztina Varga) using isotopically labelled Pγ that identified the central polycationic region of Pγ (a.a. 28-38) as the major surface of interaction with chicken cone GAFab. The second aim is to elucidate the role of the N-terminal region of cone PDE6. To address this goal, I cloned and purified chicken cone GAFab 1-458. We determined that the presence of the first ~50 amino acid residues in the N-terminus of GAFab domain is responsible for stabilizing the formation of the GAFab dimer when compared with GAFab 42-458 which primarily exists as monomer. My data showed that the presence of both the N-terminal region and binding of Pγ are responsible for enhancing the binding of cGMP affinity to GAFa domain. Using CL-MS analysis of GAFab 1-458 in conjunction with homology modeling, I generated model for the structure of chicken cone GAFab 1-458 bound to Pγ in the presence and absence of cGMP. My results unexpectedly showed that the N-terminal region of each cone PDE6 catalytic subunit fold over the surface of the GAFa domain in contrast to the extended intertwined N-terminal for rod PDE6. Moreover, the cone PDE6 N-terminal region was found to interact with the non-catalytic cGMP binding site in GAFa. Comparison of Pγ-bound GAFab (GAFab-Pγ) model with GAFab bound to both Pγ and cGMP (GAFab-Pγ-cGMP) identified structural elements undergoing conformational changes upon binding of cGMP that may be a part of the allosteric communication pathway. This study advanced our understanding of the differences in rod and cone PDE6 structure and regulation that likely contribute to differences in the electric responses of the rod and cone photoreceptors. In addition, the atomic-level knowledge of important elements in PDE6 structure, function and regulation will enable better prediction of the pathogenicity of human mutations present in the regulatory domains of GAF-containing PDEs.

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