Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy

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, multidomain 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 cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length 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.

Cobalt–Sulfur Coordination Chemistry Drives B12 Loading onto Methionine Synthase

“…A series of bacterial MTR structures obtained by cryo-EM and AlphaFold2 analyses has provided snapshots into the conformational switching between the reactivation (with cob(II)alamin bound) and resting (with MeCbl bound) states of the enzyme. 10 In the reactivation state, the AdoMet and B 12 domains are juxtaposed, positioned for methyl group transfer (Figure 1C). However, the B 12 domain, which is largely buried, is not readily accessible to MMADHC.…”

“…In our study, a truncated form of MTR, lacking the CH 3 –H 4 F domain, was used, which precluded analysis of whether AdoMet or CH 3 –H 4 F is the preferred methyl donor during cofactor loading. A series of bacterial MTR structures obtained by cryo-EM and AlphaFold2 analyses has provided snapshots into the conformational switching between the reactivation (with cob­(II)­alamin bound) and resting (with MeCbl bound) states of the enzyme . In the reactivation state, the AdoMet and B 12 domains are juxtaposed, positioned for methyl group transfer (Figure C).…”

“…The roles, if any, of the homocysteine and CH 3 –H 4 F modules in regulating cofactor transfer from MMADHC are not known. CH 3 –H 4 F binding to bacterial MTR uncaps and exposes the B 12 domain, switching the enzyme from a resting to a catalytically active conformation . While it is possible that CH 3 –H 4 F binding plays a similar role in increasing access to the B 12 domain, it does not appear to be essential for cofactor loading onto human MTR, since both the truncated variant lacking this domain and the full-length protein in the absence of CH 3 –H 4 F could be loaded with B 12 from MMADHC.…”

“…A recent structural characterization of E. coli MTR revealed that in its resting state, the B 12 domain is nestled between the CH 3 –H 4 F and homocysteine domains, while the AdoMet domain is conformationally dynamic . In the reactivation cycle, the AdoMet domain engages with the B 12 domain as seen in crystal structures of the C-terminal half of the protein .…”

“…A recent structural characterization of E. coli MTR revealed that in its resting state, the B 12 domain is nestled between the CH 3 −H 4 F and homocysteine domains, while the AdoMet domain is conformationally dynamic. 10 In the reactivation cycle, the AdoMet domain engages with the B 12 domain as seen in crystal structures of the C-terminal half of the protein. 11 The AlphaFold model of full-length MTR represents the reactivation conformation in which the AdoMet domain protects the B 12 -binding site from solvent exposure while the cap domain is disengaged 19 (Figure 1C).…”