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A Disulfide-Stabilized Conformer of Methionine Synthase Reveals an Unexpected Role for the Histidine Ligand of the Cobalamin Cofactor

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A Disulfide-Stabilized Conformer of Methionine Synthase Reveals an Unexpected Role for the Histidine Ligand of the Cobalamin Cofactor A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor Supratim Datta*, Markos Koutmos†, Katherine A. Pattridge†, Martha L. Ludwig†‡, and Rowena G. Matthews*§¶ *Life Sciences Institute, †Biophysics Research Division, and §Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-2216 Contributed by Rowena G. Matthews, January 14, 2008 (sent for review December 6, 2007) B12-dependent methionine synthase (MetH) from Escherichia coli is demethylation of CH3-H4folate separated by Ϸ50 Å (6). The a large modular protein that is alternately methylated by methyl- structure of the isolated cobalamin-binding module (residues tetrahydrofolate to form methylcobalamin and demethylated by 651–896) revealed that the cobalamin is sandwiched between homocysteine to form cob(I)alamin. Major domain rearrangements two domains: a four-helix bundle (Cap domain) lying over the are required to allow cobalamin to react with three different sub- upper (␤) face of the cobalamin and a Rossman ␣/␤ domain (Cob strates: homocysteine, methyltetrahydrofolate, and S-adenosyl-L- domain) that provides binding determinants for the lower (␣) methionine (AdoMet). These same rearrangements appear to face of the cobalamin (7). This conformation is referred to as preclude crystallization of the wild-type enzyme. Disulfide cross- Cap:Cob in Fig. 2. N␧2 of His-759 in the Cob domain is linking was used to lock a C-terminal fragment of the enzyme into coordinated to cobalt in the ␣ position. N␦1 of His-759 is also a unique conformation. Cysteine point mutations were introduced hydrogen bonded to Asp-757, which in turn is hydrogen bonded at Ile-690 and Gly-743. These cysteine residues span the cap and the to Ser-810. This set of residues, known as the ligand triad, helps cobalamin-binding module and form a cross-link that reduces the secure the histidine ligand and plays an important role in conformational space accessed by the enzyme, facilitating protein determining the distribution of conformers during catalysis (9, crystallization. Here, we describe an x-ray structure of the mutant 10). When His-759 is coordinated to the cobalt, the enzyme is fragment in the reactivation conformation; this conformation en- said to be in the His-on state, with the MeCbl cofactor exhibiting ables the transfer of a methyl group from AdoMet to the cobalamin an absorbance maxima at 525 nm (red). His-off MeCbl has an cofactor. In the structure, the axial ligand to the cobalamin, absorbance maximum at 450 nm (yellow) that can readily be His-759, dissociates from the cobalamin and forms intermodular distinguished from the His-on form, which in principle allows contacts with residues in the AdoMet-binding module. This unan- quantitation of the fraction of enzyme in the reactivation ticipated intermodular interaction is expected to play a major role conformation (9). in controlling the distribution of conformers required for the A structure of a C-terminal fragment comprising the Cob and catalytic and the reactivation cycles of the enzyme. AdoMet modules of catalytically inactive His759Gly MetH(649– 1227) in the cob(II)alamin form has also been determined (8), multimodular protein ͉ protein conformation ͉ vitamin B 12 demonstrating a dramatic rearrangement of the Cap and Cob domains that allows access of the AdoMet-binding module to the ethionine synthase (MetH) catalyses the transfer of a lower (␤) face of the cobalamin. This conformation is referred Mmethyl group to homocysteine (Hcy) to form methionine, to as AdoMet:Cob in Fig. 2. The cap is translated 25 Å from the with its cobalamin cofactor serving as an intermediary. During position it occupies in the structure of the isolated cobalamin catalysis the methylcobalamin cofactor is demethylated by Hcy domain and the corrin moiety moves away from the Cob domain to form cob(I)alamin and methionine and then remethylated by toward the AdoMet binding module. The cobalt is 2.3 Å further methyltetrahydrofolate (CH3H4folate) to form tetrahydrofolate away from C␣ of Gly-759 in the AdoMet:Cob structure than in (H4folate) and regenerate methylcobalamin (MeCbl). The the Cap:Cob structure of the isolated cobalamin domain. This MetH catalytic cycle is shown in red in Fig. 1. Under microaero- structural rearrangement, if physiologically relevant, will be philic conditions, cob(I)alamin is oxidized to the catalytically accompanied in the WT protein, whether full-length or the inactive cob(II)alamin form approximately once in every 2,000 C-terminal fragment, by bond dissociation between His-759 and turnovers (1). To return Escherichia coli MetH to the catalyti- cobalt, thus leading to the formation of a His-off cob(II)alamin cally competent methylcobalamin form, the cofactor is reduced species. by flavodoxin and methylated by S-adenosyl-L-methionine CHEMISTRY The aforementioned molecular rearrangement is only one of (AdoMet) (2, 3), a reactivation sequence shown in blue in Fig. the several rearrangements that MetH undergoes: each of the 1. MetH can thus access two alternative methyl donors, CH - 3 methyl transfer reactions shown in Fig. 1 occurs at a different H4folate and AdoMet, as well as one methyl acceptor, Hcy. To prevent futile cycling of cob(I)alamin during the catalytic cycle, the enzyme discriminates against AdoMet; during the reactiva- Author contributions: S.D. and M.K. contributed equally to this work; S.D., M.K., M.L.L., and tion sequence, the enzyme similarly discriminates against R.G.M. designed research; S.D. and M.K. performed research; S.D., M.K., K.A.P., and R.G.M. CH3H4folate (4). These studies have shown that the cob(I)- analyzed data; and S.D., M.K., and R.G.M. wrote the paper. alamin form of the enzyme is unable to switch between the The authors declare no conflict of interest. BIOCHEMISTRY conformations necessary for catalysis and reactivation. How- Freely available online through the PNAS open access option. ever, the structural basis for such discrimination remained Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, unknown. www.pdb.org (PDB ID code 3BUL). MetH is a modular enzyme (Fig. 2) with four functional units ‡Deceased November 27, 2006. that bind Hcy, CH3-H4folate, cobalamin, and AdoMet (5). The ¶To whom correspondence should be addressed at: 4002 Life Sciences Institute, 210 crystal structures of the N-terminal modules of MetH from Washtenaw Avenue, Ann Arbor, MI 48109-2216. E-mail: [email protected]. Thermotoga maritima and the C-terminal modules from E.coli This article contains supporting information online at www.pnas.org/cgi/content/full/ have been solved (6–8). The Hcy- and folate-binding modules 0800329105/DC1. are (␤␣)8 barrels, with the sites for methylation of Hcy and © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800329105 PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4115–4120 Downloaded by guest on October 1, 2021 Fig. 2. Cartoon of a minimal set of conformations for the methylcobalamin forms of MetH(2–1227) (Upper) and MetH(649–1227) (Lower). The Hcy- binding module is shown in green, the folate-binding module in light yellow, Fig. 1. The catalytic (red) and reactivation (blue) cycles of E. coli MetH. the cobalamin-binding domain in red, the Cap domain in dark yellow, and the During catalysis the cobalamin cofactor is alternately methylated by CH3- AdoMet-binding domain in blue. The corrin ring of the cobalamin is repre- H4folate and demethylated by Hcy. The cob(I)alamin form of the enzyme is sented by the square, and the vertical line below it indicates the His-on occasionally oxidized to form the inactive cob(II)alamin form. Return of this conformation of the Hcy:Cob, Fol:Cob, and Cap:Cob conformations. In the species to the catalytic cycle involves reduction with electrons derived from AdoMet:Cob conformation, the histidine is displaced and the corrin moves reduced flavodoxin and methylation with a methyl group derived from away from the cobalamin-binding domain to assume a His-off conformation. AdoMet. selecting appropriate residues are described in ref. 12. The substrate-binding domain and requires a different arrangement residues chosen were in different domains of the cobalamin of modules (Fig. 2). During the catalytic cycle, the enzyme module, with Ile-690 in the Cap domain and Gly-743 in the linker oscillates between a conformation with the CH3-H4folate- connecting the Cap and Cob domains. This disulfide cross-link binding and Cob domains positioned for methylation of cobal- is expected to tether the enzyme in the AdoMet:Cob confor- amin (Fol:Cob) and a conformation with the Hcy and Cob mation because it shortens the nine residue linker region be- domains positioned for demethylation (Hcy:Cob). Recent stud- tween the Cap and Cob domains by four residues and is ies have examined the distribution of molecular species in the positioned so as to restrict movement of the Cap relative to the MeCbl form of MetH and shown that the enzyme exists as an Cob domain. To eliminate the possibility of intramolecular ensemble of interconverting conformations. The distribution of disulfide linkages between the cysteine mutants and the native states at equilibrium depends on the oxidation and ligation state cysteines at positions 772, 1042 and 1142 of MetH(649–1227), of the cobalamin, on the temperature, and on the concentrations the two cysteine mutations had to be introduced at positions with of substrates or products (9, 11). C␣ separations of at least6Åormore from each of the native In this study, we have used disulfide cross-linking to lock the cysteines. C-terminal fragment of WT MetH into the reactivation confor- mation before crystallization. Specifically, we engineered unique Expression and Characterization of I690C/G743C MetH(649–1227).
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