Structure and Mechanism of the Glucose-6-Phosphatase Catalytic Subunit 1

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Principal Investigator: Derek P Claxton
Organization: VANDERBILT UNIVERSITY
Fiscal Year: 2024
Award: $338,889
Funding agency: National Institute of General Medical Sciences

Project Summary
 This proposal requests support for investigating the structure and enzymology of glucose-6-phosphatase
catalytic subunit 1 (G6PC1), an integral membrane protein of the phosphatidic acid phosphatase type 2 (PAP2)
superfamily expressed in the ER that catalyzes glucose production in the liver. Unregulated G6PC1 activity
contributes to diabetes pathology by driving increases in hepatic glucose production and fasting blood glucose.
In contrast, mutations in G6PC1 that impair activity cause glycogen storage disease type 1a (GSD type 1a),
which is characterized primarily by severe hypoglycemia. Despite its physiological relevance to glucose
metabolism, the structural basis of catalysis as well as the impairment caused by disease-linked mutations is
poorly understood. This application outlines an experimental plan encompassed within three specific aims that
integrates complementary biophysical and computational techniques to study the mechanistic structural biology
of mouse G6PC1 (mG6PC1), a stable G6PC1 ortholog, in unprecedented detail. The research plan is facilitated
by published methodological advancements developed in the PI’s laboratory that support purification of
functional mG6PC1 from Sf9 cell membranes. The scientific approach capitalizes on preliminary 3D models
predicted within the AlphaFold2 (AF2) computational framework as templates for experimental design. The
cornerstone of this proposal is the application of double electron electron resonance (DEER) spectroscopy
between nitroxide spin labels introduced into the mG6PC1 sequence to establish the veracity of computational
models, obtain restraints for structural refinement and to describe the conformational dynamics of the catalytic
cycle. The relative stability of these intermediates will be assessed in the background of mutants known to cause
GSD type 1a. Informed by the spectroscopic analysis, cryogenic electron microscopy single particle analysis will
be used to build high resolution models of stable catalytic intermediates. The experimental approach is grounded
in the expertise of the PI and the team of established collaborators in the molecular biology of G6PC1 and the
biophysical analysis of membrane proteins employing the described toolkit. A working mechanistic model has
been derived from pilot spectroscopic studies indicating that mG6PC1 operates by a substrate-dependent
conformational equilibrium between “resting” and “active” states mediated by relative rearrangement of a unique
ancillary motif of transmembrane helices attached to the conserved PAP2 domain. Molecular dynamics
simulations in conjunction with a robust in vitro platform for screening expression and activity have uncovered
multiple modes of catalytic inhibition induced by GSD type 1a variants found in the active site. The research
strategy will extend these findings beyond the active site into other hot spots for missense mutations found in
the supporting loops and transmembrane helices. Interpreted within the context of high-resolution molecular
models, the results of this work will uncover the orchestrated pattern of conformational changes underpinning
catalysis as well as the thermodynamic and kinetic basis of inhibition induced by disease-linked mutations.

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