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Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron 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, 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.

SUBMITTER: Watkins MB

PROVIDER: S-EPMC9934640 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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

Watkins Maxwell B MB Wang Haoyue H Burnim Audrey A Ando Nozomi N

bioRxiv : the preprint server for biology 20230212

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 preve ...[more]

PMID: 36798380

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Project description:Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D2O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10(-4) cm(3)mol/g(2) for Mb in phosphate buffer, 1.6 ×10(-4) cm(3)mol/g(2) for BPTI in phosphate buffer and 9.2 ×10(-4) cm(3)mol/g(2) for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.

Dataset Information

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

Publications

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

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