Factors modulating conformational equilibria in large modular proteins: A case study with cobalamin-dependent methionine synthase

Abstract

In the course of catalysis or signaling, large multimodular proteins often undergo conformational changes that reposition the modules with respect to one another. The mechanisms that direct the reorganization of modules in these proteins are of considerable importance, but distinguishing alternate conformations is a challenge. Cobalamin-dependent methionine synthase (MetH) is a 136-kDa multimodular enzyme with a cobalamin chromophore; the color of the cobalamin reflects the conformation of the protein. The enzyme contains four modules and catalyzes three different methyl transfer reactions that require different arrangements of these modules. Two of these methyl transfer reactions occur during turnover, when homocysteine is converted to methionine by using a methyl group derived from methyltetrahydrofolate. The third reaction is occasionally required for reactivation of the enzyme and uses S-adenosyl-L-methionine as the methyl donor. The absorbance properties of the cobalamin cofactor have been exploited to assign conformations of the protein and to probe the effect of ligands and mutations on the distribution of conformers. The results imply that the methylcobalamin form of MetH exists as an ensemble of interconverting conformational states. Differential binding of substrates or products alters the distribution of conformers. Furthermore, steric conflicts disfavor conformers that juxtapose a methyl group on substrate with one on methylcobalamin. These results suggest that the methylation state of the cobalamin will influence the distribution of conformers during turnover.

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

“…Although interactions between the active sites and more distant residues might be revealed by further studies (43), current observations from the structure fit the earlier view that changes in the distribution of conformations (Fig. 1B) are largely governed by local interactions (11). The modeling studies (Fig.…”

“…The factors that govern the distribution of these species are now partly understood. The oxidation and ligation states of cobalamin strongly influence the population of the accessible domain arrangements (11,42). Recent studies of MetH from E. coli have demonstrated that substrates and products can also alter the relative stabilities of the different conformations (11).…”

“…Intact MetH appears to sample a limited number of domain arrangements that are not vastly different in energy (11) (Fig. 1B).…”

“…Recent studies have demonstrated that the enzyme exists as an ensemble of conformations with equilibria dependent on the oxidation and ligation state of the cobalamin and on the concentrations of substrates or products (11). At a minimum, the ensemble in the intact enzyme comprises four species in which the cobalamin-binding domain is positioned to interact with one or another of its four possible partners (Fig.…”

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

“…There is precedent in the Cbl enzyme literature for a large-scale conformational change upon substrate binding (32,37,39). For methylcobalamin-dependent methionine synthase, the enzyme exists as an ensemble of conformational states that interconvert upon substrate or product binding (39).…”