Characterizing E. coli Phosphoenolpyruvate Carboxykinase Conformational States through Small Angle X-Ray Scattering

Abstract

The conformations of an enzyme are directly related to function: during catalysis, conformational changes are required to bring catalytic sidechains into position for the reaction. Ligand binding often results in gross conformational changes as well. While X-ray crystallography provides atomic resolution enzyme structures, they are static conformations captured in the crystallographic state. Deviation of crystal structures from actual solution state conformation can arise from crystal packing as well as the non-physiological conditions required for crystal growth. Combining atomic resolution data from crystallography with solution small angle X-ray scattering (SAXS) data allows us to observe small conformational changes at resolutions greater than SAXS alone under conditions that are a more accurate representation of a protein's native environment. Phosphoenolpyruvate carboxykinase (PCK) is a key metabolic enzyme responsible for catalyzing the first committed step of gluconeogenesis. It is a bilobate enzyme with the active site located in the cleft between the two domains. From crystallographic studies, substrate binding leads to domain rotation and cleft closing, and a 10-residue cap closes over the cleft. SAXS experiments on apo PCK suggests that the cap is in a closed state and the domains do not stay in a fully open state. Certain active site mutants of PCK show drastically different behavior in solution, including a constitutively open mutant that showed a different SAXS scattering profile. Addition of ATP to WT PCK led to both cleft and cap closing observable by SAXS. We also present a tool for high throughput comparison of SAXS profiles. From these results, it is evident that when SAXS experiments are combined with atomic models, relatively small conformations can be captured. This extends SAXS capabilities beyond its current routine use of generating low resolution molecular envelopes.