Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy
Year: 2023
Type: article
Abstract: Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH 3 -H 4 folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S -adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multi-domain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a “divide and conquer” approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryo-electron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present the first structural description of MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH 3 -H 4 folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.
Institutions: Princeton University, Cornell University
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: hybrid
Funders: National Science Foundation, Empire State Development Corporation, Williams College, Princeton University, Johns Hopkins University, University of Texas Southwestern Medical Center, National Institutes of Health, Cornell Center for Materials Research, National Institute of General Medical Sciences, Division of Materials Research, Common Fund
Awards: Structural Characterizations of Transient and Heterogeneous Protein Complexes, G3495479163, OPERATION OF the NSF Center for High Energy X-ray Science (NSF-CHEXS), G4582736256, G5860078510, G5903464632, Structural Characterizations of Transient and Heterogeneous Protein Complexes, G6971479070, G7051197921, G8142210830, NCCAT: National Center for CryoEM Access and Training, Lab Source for Small/Wide Angle X-ray Scattering Experiments, G9000339200, Protein Allostery and Catalysis Beyond Bragg Diffraction, MacCHESS Synchrotron Source for Structural Biology
OpenAlex ID: W4381327660