Date of Award

Spring 2022

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Rick H Cote

Second Advisor

Feixia Chu

Third Advisor

David Plachetzki

Abstract

The photoreceptor phosphodiesterase (PDE6) plays an important role in the G-protein coupled visual signaling pathway which uses cGMP as a second messenger to convert light stimuli into electrical signals. PDE6 is a tetrameric peripheral membrane protein consisting of two catalytic subunits and two inhibitory subunits and is localized to the outer segment membranes of rod and cone photoreceptors. Mutations in this enzyme are one cause of retinitis pigmentosa and other retinal degenerative diseases resulting in blindness or visual dysfunction that lack adequate therapeutic intervention due to inadequate knowledge of PDE6 structure and regulation PDE6 is tightly regulated in the nonactivated state, as well as during activation and deactivation of the visual signaling pathway. In the nonactivated state, the rod PDE6 catalytic dimer (consisting of the Pα and Pβ catalytic subunits) is inhibited by a pair of identical inhibitory subunits (Pγ) to form the PDE6 holoenzyme (Pαβγγ). Activation of PDE6 results from displacement of the Pγ subunit by the light-activated G protein alpha-subunit (Gtα). Deactivation of PDE6 is the result of the GTPase activity of Gtα which is aided by a GTPase accelerating complex consisting of the Regulator of G Protein Signaling 9 (RGS9-1), the obligate dimer to RGS9-1, Gβ5L, and the RGS9-1 anchoring protein (R9AP). Together this inactivation complex allows PDE6 to return to the nonactivated conformation. The hypotheses of my research are: (1) silica particles encased by large unilamellar phospholipid vesicles will mimic the photoreceptor membrane and provide a surface suitable for enhancing the interactions of PDE6 and Gtα as well as the proteins involved in the deactivation complex; (2) one Gtα molecule binds to each PDE6 catalytic domain and induces a large conformational change in the inhibitory Pγ subunit; (3) interaction of RGS9-1 with Gtα will induce changes in the interaction surface between activated Gtα and PDE6, allowing Pγ to resume the conformation which inhibits PDE6 activity. The first aim of my research is to establish a methodology to study PDE6 and its associated complexes in a system that mimics the rod outer segment. In order to achieve this, a protocol for encasing silica particles in large unilamellar phospholipid vesicles (called “lipobeads”) was developed. This methodology not only allowed for an increase in the extent of Gtα activation when compared to PDE6 in solution, but also allowed for study of membrane-attached PDE6 and Gtα at concentrations that more closely mimic those observed in the rod outer segment. The second aim of my research is to characterize the structure of membrane-attached PDE6 in its nonactivated state and in the fully activated state upon binding of Gtα. This was achieved using chemical crosslinking and mass spectrometry in conjunction with a computational modeling program called the Integrative Modelling Platform. In the nonactivated state, it was observed the Pγ has significant interaction with the regulatory GAFa domain as well as the catalytic domain of Pαβ while displaying a less well defined structure in the central cationic region of Pγ. Upon activation, two Gtα are bound to specific docking sites on PDE6 resulting in the displacement of Pγ from both catalytic domains as well as a predicted shift of Pγ away from GAFa. The third aim of my research is to understand the sequential activation mechanism of PDE6 by Gtα. Chemical crosslinking and mass spectrometry was again used in order to characterize the structures of PDE6 with a sub-stoichiometric amount of Gtα as well as a slight stoichiometric excess of Gtα (0.4:1 and 3:1 Gtα:PDE6, respectively). In the case of the stoichiometric excess, a high molecular weight cross-linked band on SDS-PAGE indicative of two Gtα bound to PDE6 was structurally analyzed; the sub-stoichiometric condition resulted in a single Gtα bound species which was also analyzed. Comparisons were also made between the inactive (Gtα-GDP) and activated (Gtα*-GDP-AlF4-) states. This work showed that when two activated Gtα* molecules were bound to PDE6 both Gtα subunits were associated with the catalytic domains of PDE6. When Gtα was present at sub-stoichiometric levels relative to PDE6, a single docking site was identified in proximity to the GAFb domains of PDE6. The inactive state of Gtα (Gtα-GDP) also was capable of binding PDE6 but bound only to the GAFb domains. Measurements of PDE catalytic activity established two Gtα-GDP-AlF4- molecules were able to produce significant activation of PDE6, whereas the sub-stoichiometric condition (0.4 Gtα per PDE6) did not produce activity above basal levels. These results indicate that the binding of a single Gtα is not sufficient to stimulate activity of PDE6. The final aim of my research is to establish a methodology for the study of the deactivation complex of PDE6. To achieve this aim, lipobeads were used in order to anchor the integral membrane protein R9AP to produce “proteolipobeads”. This membrane-embedded R9AP preparation was then able to bind the RGS9-1/Gβ5L without affecting the ability of PDE6 and Gtα to also bind to the proteolipobeads. Chemical crosslinking and mass spectrometry analysis confirmed that all of the proteins were present on the membrane and in close enough proximity to allow future analysis of the PDE6 inactivation complex.

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