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

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 conformation before crystallization. Specifically, we engineered unique Expression and Characterization of I690C/G743C MetH(649–1227). The cysteine residues positioned to form a disulfide that would span mutant enzyme was overexpressed and purified to Ͼ95% purity the Cap and Cob domains of MetH. The I690C/G743C as judged by electrophoretic analysis under denaturing condi- MetH(649–1227) becomes locked in the reactivation conformations and is isolated as a mixture of cob(II)alamin and aquaco- tion. The axial ligand to the cobalamin moiety, His-759, is balamin forms. Reductive methylation in an electrochemical cell dissociated from the cobalamin. Relative to the distance from C␣ in the presence of AdoMet converted the protein to the meth- of His-759 to cobalt in the Cap:Cob conformation (7), the cobalt ylcobalamin form. The methylated enzyme is very light sensitive, is displaced by 2.9 Å in the reactivation conformation. Unex- so to prevent photolysis of the methyl-cobalt bond, all subse- pectedly, the displaced His-759 is now involved in intermodular quent experiments were performed in the dark. Whereas WT contacts with the AdoMet module. The N␧2 nitrogen of His-759 MetH(649–1227) at room temperature gave a spectrum consis- interacts with the AdoMet domain directly through Asp-1093 tent with His-on MeCbl, with an absorbance maximum at 525 nm Ϫ1 Ϫ1 and via a water molecule to Glu-1069. These interactions would (␧525 ϭ 9,400 M cm ), the spectrum of I690C/G743C be expected to stabilize the AdoMet:Cob conformation, and may MetH(649–1227) showed a maximum at 452 nm and an ␧452 of Ϫ1 Ϫ1 rationalize the failure of the cob(I)alamin form of MetH to 10200 Ϯ 100 M cm . This blue shift of the ␭max compared with interconvert between AdoMet:Cob and Fol:Cob conformations WT MetH indicates that I690C/G743C MetH(649–1227) is of the enzyme. mostly His-off.

Results and Discussion I690C/G743C MetH(649–1227) Is Found in both His-On and -Off States. Introduction of a Disulfide Cross-Link to Reduce the Conformational The methylcobalamin form of both WT and I690C/G743C Flexibility of MetH. A remaining challenge for understanding the MetH(649–1227) showed temperature-dependent changes in complex reaction sequence of MetH is to understand how the absorbance consistent with an interconversion between a His-off domains interact and rearrange during the reaction cycle. Crystal species at high temperature and a His-on species at low tem- structures of the full length enzyme in the different conforma- peratures, as shown in Fig. 3. These changes were used to tions are essential for achieving this goal. Unfortunately, crys- calculate equilibrium constants [K ϭ (% His-off)/(% His-on)]. tallization of the full-length enzyme has been unsuccessful to From the plot of ln K vs. TϪ1, we calculated ⌬H ϭ 27.5 kcal/mol date, presumably due to the conformational flexibility of MetH. and ⌬S ϭ 83.2 cal/mol/K for the interconversion between His-on A different strategy was thus required to tackle this problem, and and His-off species of WT MetH(649–1227). These values we chose originally to implement it in the C-terminal fragment yielded a ⌬G of ϩ 1.73 kcal/mol at 37°C, where the positive value of MetH. We used an intramodular disulfide cross-link to reduce indicates a preference to remain in the His-on conformation. A the conformational flexibility of MetH. value for ⌬G of ϩ 2.55 kcal/mol was determined previously for The structure of the C-terminal MetH fragment in the the full-length WT enzyme (11). This more positive value is AdoMet:Cob conformation was used to select residues suitable probably due to the larger number of His-on conformations in for mutation to cysteines (8). The stereochemical criteria for the full-length enzyme (Fig. 2). Similar experiments were per-

4116 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800329105 Datta et al. Downloaded by guest on October 1, 2021 Fig. 4. A ribbon drawing of I690C/G743C MetH(649–1227). The activation domain (blue) caps the corrin ring and interacts with the cobalamin-binding Fig. 3. The effect of temperature on the absorbance of WT MetH(649–1227) domain (red) and the four-helix bundle or cap (yellow). The cysteine residues (A and B) and I690C/G743C MetH(649–1227) (C and D). The spectra were are shown in space ﬁlling rendition, and the cobalamin cofactor (CPK coloring) recorded in 10 mM potassium phosphate buffer, pH 7.2, after equilibration for is rendered in stick. 2 min at 10°C (purple), 15°C (deep blue), 20°C (light blue), 25°C (green), 30°C (light green), and 37°C (orange). Arrows indicate the direction of absorbance changes as the temperature is increased. The equilibrium constant, K ϭ duced cysteines, we performed disulfide mapping experiments (%His-off/%His-on), was calculated at each temperature to create van’t Hoff based on the method of Wu et al. (14) and Hondorp et al. (15). plots, shown on the right. Thermodynamic parameters were calculated as The data are shown in supporting information (SI) Table 2 and described in Methods. SI Fig. 7. The five cysteines of the mutant protein are located at residues 690, 743, 772, 1042, and 1142, and of these, 690 and 743 ⌬ are predicted to form a disulfide bond. Mass spectrometric formed with the I690C/G743C double mutant. H was calcu- analysis of the cyanylated and cleaved I690C/G743C MetH(649– lated to be 6.2 kcal/mol and ⌬S ϭ 26.9 cal/mol/K, yielding a ⌬G Ϫ ⌬ 1227) allowed the identification of all but one of the expected of 2.1 kcal/mol at 37°C. The negative value of G seen for the peptides. Repeated attempts by mass spectrometry proved un- mutant reflects the preference for the enzyme to be in the successful in identifying peptide 1–772, which would contain the His-off form, presumably attributable to the stabilizing effect of disulfide cross-link, although peptides indicating cleavage at the disulfide cross-link on this species. cysteines 772, 1042, and 1142 were observed. The absence of the In contrast, the aquacobalamin form of I690C/G743C disulfide cross-link between Cys-690 and Cys-743 would have MetH(649–1227) is red at all temperatures, consistent with the 3ϩ given rise to two extra peptide fragments containing residues very high affinity of Co in aquacobalamin for a coordinated 1–689 and 690–771, but no evidence was found for the presence base in the lower axial position (13). Surprisingly, the cross- of these peptide fragments. Thus, the evidence from DTNB linked mutant structure can assume a His-on conformation (see titrations and mass spectrometry suggests the presence of a below). disulfide bond between cysteines 690 and 743. Thiol Quantitation. The free thiols were quantitated by assaying Overall Structure of I690C/G743C MetH(649–1227). We obtained with DTNB. WT MetH(649–1227) enzyme contains a total of crystals of the mutant MetH at 37°C, where the enzyme was three free thiols (Table 1), as expected from the amino acid mostly in the His-off conformation. The overall architecture of sequence. The I690C/G743C enzyme contains a total of five I690C/G743C MetH(649–1227) is shown in Fig. 4. Crystallo- cysteines, but only three free thiols were found by DTNB graphic information as well as refinement statistics are provided CHEMISTRY titration, indicating the presence of a disulfide cross-link. in SI Table 3. The enzyme was crystallized in the activation To verify that the intramodular disulfide involved the intro- conformation where the helical cap region and the cobalamin binding module are adjoined to each other with the AdoMet Table 1. Sulfhydryl group quantitation by DTNB titration binding module on top of the cobalamin moiety. Cys-690 is positioned at the beginning of helix Ia3 in the cap domain (7) and Cysteines based on Cys-743 is in the linker region connecting the Cap and Cob DTNB domains and is located in a flexible region of the protein. This

Enzyme Sequence assay Disulﬁdes flexibility is reflected in the poor electron density corresponding BIOCHEMISTRY to this loop in both the His759Gly mutant structure previously WT MetH(649–1227) determined (8) and in the present disulfide cross-linked struc- Ϯ Methylcobalamin form 3 2.9 0.1 0 ture. Consequently, we cannot unequivocally model the two Ϯ Aquacobalamin form 3 3.0 0.2 0 cysteine side chains in the observed electron density. However, I690C/G743C MetH(649–1227) whereas the position of the side chains of the residues in that area Ϯ Methylcobalamin form 5 2.7 0.1 1 cannot be well defined, the backbone is clearly defined and the Aquacobalamin form 5 2.9 Ϯ 0.1 1 C␣OC␣ distance of 5.4 Å is consistent with a disulfide. The The titrations were performed at room temperature as described in electron density in this region is shown in SI Fig. 8. Moreover, Methods. as predicted, the distances between the native and the engi-

Datta et al. PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4117 Downloaded by guest on October 1, 2021 Fig. 5. A comparison of the active site of I690C/G743C MetH(649–1227) in the His-off conformation (Right) and the cobalamin binding module of WT MetH (651–896) (Left), in the His-on conformation. The cobalamin cofactor undergoes signiﬁcant movement relative to its position in the isolated cobalamin binding module; note in particular the tilting of the equatorial plane of the corrin ring in the structure of I690C/G743C MetH(649–1227), which moves the cobalt away from C␣ of His-759 by 2.9 Å. The side chain of His-759 undergoes a dramatic reorientation, whereas the positions of the distal residues comprising the catalytic triad, Asp-757, and Ser-810 are relatively unchanged between the two conformations.

neered cysteines are long enough to preclude disulfide bond a ‘‘His-off state’’ was hitherto unknown. The presence of this formation. Thus, although the presence of the disulfide bond ligand in our structure provides us with the opportunity to assess cannot be unambiguously demonstrated by crystallography, the the differences as well as the similarities between the His-off and evidence based on DTNB titrations, mass spectrometry, ther- His-on states where the latter is represented by the structure of modynamic parameters, and the overall conformation of the WT MetH (651–896) (7). As shown in Fig. 5, a very substantial enzyme in the structure, is consistent with the presence of a rearrangement of His-759 in the His-off conformation is evident Cys-690–Cys-743 disulfide cross-link. when compared with the structure of the isolated cobalamin The structure of I690C/G743C MetH(649–1227) is very sim- fragment in the His-on Cap:Cob conformation. In the His-off ilar to that of the previously determined His759Gly MetH(649– state His-759 not only moves away with respect to Co as evident 1227) (a comparison of the two structures is shown in SI Fig. 9). by the C␣ displacement of 2.9 Å, but the histidine ring also When the two structures are compared, each of the individual rotates and faces away from the Co atom. However the differ- domains is very similar, and they move as rigid bodies relative to ences between the His-on and His-off states, based on these two one another. The cobalamin-binding domain in the disulfide structures, appear to be only in the His-759 and cobalamin cross-linked structure is now closer to the AdoMet module and positions. Although the cobalamin and histidine ring seem to be the cofactor, although the position of the corrin ring relative to moving away from each other in going from the His-on to the the AdoMet module is basically unchanged. As will be discussed His-off form, other residues in the cobalamin domain, including below, the closer approximation of the AdoMet and Cob do- the other two residues of the catalytic triad (Ser-810 and mains may be a result of the new intermodular contacts made by Asp-757), align well with each other. His-759. Although His-759 is not bound to cobalt, it still interacts with cobalamin through a hydrogen bond between N␦1 and one of the The Cobalamin has Photolyzed. No electron density was observed propionamide side chains. More importantly, His-759 makes at either face of the cobalamin moiety near the cobalt atom, specific contacts with the AdoMet module. In I690C/G743C indicating the absence of the methyl group in the crystal MetH(649–1227) His-759 forms a hydrogen bond with Asp-1093 structure and a resulting four-coordinate cobalt species (Fig. 5). and a water mediated hydrogen bond to Glu-1069 (Fig. 6). These SI Fig. 10 shows the electron density. The result was unexpected two residues are both located in the AdoMet-binding module of because the crystallization trays were set up with the MeCbl form of I690C/G743C MetH(649–1227). Although care was taken to MetH. Asp-1093 is a highly conserved residue among all MetH expose the enzyme and the crystal to as little light as possible, homologues, whereas the consensus sequence for MetH indi- the I690C/G743C MetH(649–1227) is more susceptible to pho- cates a polar residue at the position of Glu-1069. Reduction of tolysis than the WT enzyme. The demethylation was possibly due His-on cob(II)alamin to His-off cob(I)alamin in WT MetH to photoreduction during preparation of the crystals and/or occurs with uptake of a proton, presumably on His-759 (20). The photoreduction due to exposure to high-energy x-ray to form interactions shown here are consistent with protonation of ␧ cob(II)alamin, as previously observed in other determinations of His759N 2, thus identifying Asp-1093 as a potential proton the structures of cobalamin-containing enzymes (16–19). Mag- donor. Thus, this interaction may result in the formation of a hydrogen bond between members of an ion pair. Such Hisϩ– netic circular dichroism studies of the cob(II)alamin formed by Ϫ photolysis of methylated I690C/G743C MetH(649–1227) in a Asp hydrogen bonds may contribute as much as 3–5 kcal/mol rigid glass at 77 K has established that the cobalamin is four- of stability (21). coordinate (unpublished data obtained in collaboration with This intermodular interaction is an unanticipated finding and Matthew Liptak and Thomas Brunold, University of Wisconsin, could provide a structural basis for the inability of the cob(I)- Madison). alamin form of MetH to interconvert between catalytic and reactivation conformations. When the His-759 and Gly-759 Intermodular Constraints in the His-Off Conformation. In the previ- forms of the reactivation conformation are compared, a major ously reported structure of H759G MetH(649–1227) in the difference is the intermodular bonding between His-759 and reactivation conformation (AdoMet:Cob), the critical His-759 Asp-1093, and the water-mediated interaction between His-759 residue was absent, so the position of this important ligand, when and Glu-1069. These intermodular interactions should signifi- not bound to Co, and its potential role to the enzyme while in cantly stabilize the His-759 enzyme in the reactivation confor-

4118 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800329105 Datta et al. Downloaded by guest on October 1, 2021 (Amersham Biocsciences, Uppsala, Sweden) charged with nickel sulfate and was equilibrated with 500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). The column was washed with four column volumes of this buffer and four volumes of 20 mM imidazole/500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). MetH(649–1227) was eluted with 200 mM imidazole/500 mM sodium chloride/20 mM sodium phosphate (pH 7.3). The enzyme was then exchanged into 10 mM potassium phosphate (pH 7.2) and loaded on a Mono Q 16/10 column (Amersham Biosciences) equilibrated with the same buffer. The protein was eluted with a 300-ml linear gradient between 10 and 400 mM potassium phosphate buffer. The colored fractions were pooled together, concentrated in an Amicon concentrator and exchanged into 10 mM potassium phosphate buffer, pH 7.2. Protein concentration was calculated by using Ϫ1 Ϫ1 the absorbance of the bound MeCbl cofactor (␧524 ϭ 9400 M cm ). The yields ranged from 7 to 9 mg of purified MetH(649–1227) per liter of cell culture. The I690C/G743C double mutant was introduced into the pVB8 vector using the QuikChange site-directed mutagenesis kit (Stratagene). The primers were obtained from Invitrogen. The sequence of I690C/G743C MetH(649–1227) was confirmed by complete sequencing at the Biomedical Research Core Facility of Fig. 6. Intermodular interactions involving His-759 in the His-off state. N␧2 the University of Michigan. I690C/G743C MetH(649–1227) was purified as of His-759 interacts with the AdoMet module directly through a hydrogen described above for the WT fragment. Protein concentration was calculated bond to Asp-1093 and via a water-mediated hydrogen bond to Glu-1069. N␦1 by using the absorbance of the bound His-off MeCbl cofactor (␧452 ϭ 10200 of His-759 forms a hydrogen bond with the amide of the propionamide side MϪ1 cmϪ1) after the protein was reductively methylated in the presence of chain of ring B of the cobalamin (data not shown). AdoMet (23).

Reduction, Methylation, and Photolysis of Cobalamin. I690C/G743C MetH(649– mation and prevent interconversion to the catalytic conforma- 1227) was converted to the MeCbl form by reductive methylation in an tion while the cobalamin is in a His-off state [e.g., cob(I)alamin]. electrochemical cell using AdoMet as the methyl donor (23). The cob(II)alamin A low-resolution (3 Å) structure of the aquacobalamin form form of MetH was formed by the photolysis of MeCbl MetH as described in ref. of I690C/G743C MetH(649–1227), which is always in the red 23. His-on state, confirms that the enzyme is indeed in the AdoMet: Cob conformation (data not shown). In this structure, the Crystallization and Cryoprotection. The 65-kDa I690C/G743C MetH(649–1227) fragment in 50 mM Tris buffer at pH 7.2 was concentrated to Ϸ12 mg/ml. histidine has rotated back toward the cobalt of the cobalamin Crystals were grown by slow evaporation in microbatch plates under oil (3:1 and is now close enough to serve as a ligand and the Cob domain mixture of paraffin and silicon oil) by mixing 2 ␮l of protein with 2 ␮lofa has moved closer to the cobalamin. After methylation by solution that consists of 0.2 M potassium nitrate and 20% (wt/vol) PEG3350. AdoMet, the enzyme might transiently convert to the His-on The crystallization trays were kept at 37°C for 1 week before they were species in this conformation, weakening the intermodular inter- transferred to 22°C, where they were kept until crystals were harvested. actions and allowing the enzyme to convert to a catalytic Crystals were transferred to a solution of 18% (wt/vol) PEG3350, 0.2 M conformation. It will now be of great interest to ascertain potassium nitrate, and 15% propylene glycol in 25 mM Tris buffer at pH 7.0 for whether similar intermodular interactions between His-759 and a few minutes before they were flash frozen. the Fol module in the His-off cob(I)alamin formed by demethylation during catalytic turnover also serves to lock the enzyme Data Collection and Structure Determination. Diffraction data were collected at GM/CA-CAT (Advanced Photon Source, Argonne National Laboratory, Ar- in the Fol:Cob conformation, preventing access of the cobalamin gonne, IL) on a Mar300 detector and processed with XDS (24). The crystal to AdoMet and ensuring its access to CH3-H4folate. structure of H759G MetH(649–1227) from E. coli (PDB ID 1K7Y) was used as a search model after the removal of the cobalamin cofactor, water molecules, Conclusions and residues from flexible regions with high B factors. Phases were obtained In summary, we solved a structure of a C-terminal fragment of by molecular replacement with the program EPMR (25). Initial structure methionine synthase that was stabilized by disulfide cross- refinements with CNS included simulated annealing in torsional space, coor- linking. The cross-linking significantly reduced the conforma- dinate minimization and restrained individual B-factor adjustment with max- tional flexibility of this fragment, favoring the His-off reactiva- imum-likelihood targets (26). No sigma(F) data cutoff was used in refinement. tion conformation by Ϸ3.9 kcal/mole. The His-off state of A disulfide restraint between Cys-690 and Cys-743 was introduced toward the latter stages of refinement. A final restrained refinement using isotropic B His-759 in the reactivation conformation is stabilized by its factors with REFMAC5 was implemented (27). Model modification was per- intermodular interactions with the AdoMet binding module. The formed with XtalView/Xfit (28). The geometric quality of the model and its CHEMISTRY movement required to dissociate the histidine is limited to the agreement with the structure factors was assessed with PROCHECK and side chain of this residue, with the other catalytically important SFCHECK, respectively (29, 30). Figures were generated by using Pymol (31). residues in the cobalamin-binding domain being unaffected. Determination of Cobalamin Extinction Coefficients and Concentrations. The Methods extinction coefficients of the enzyme-bound cofactor species of the Expression and Purification of MetH(649–1227) and I690C/G743C MetH(649– MetH(649–1227) and I690C/G743C MetH(649–1227) were determined as de- 1227). Amino-terminal His6-tagged E.coli WT MetH(649–1227) was generated scribed for the MeCbl-bound state of full-length WT MetH (32). by Vahe Bandarian (University of Arizona, Tucson, AZ) from pMMA11 and

pT7-H plasmids by using restriction digestion and ligation. The resultant Determination of Thermodynamic Parameters Associated with the His-On/His- BIOCHEMISTRY plasmid pVB8 enables the expression of His-tagged C-terminal MetH. The Off Conversion. MetH(649–1227) (4–6 ␮M) in 50 mM potassium phosphate enzyme was expressed in cells of E.coli Hms174(DE3) (Novagen) containing the buffer (pH 7.2) was placed in a sample cuvette in a dual beam Varian Cary Bio pVB8 plasmid. Bacteria were grown at 37°C and harvested as described in ref. 300 UV-Vis thermostated spectrophotometer and an equal amount of buffer 22. Cells were resuspended to a density of 0.5–0.75 g of cells per ml in 20 mM was added to the reference cuvette. The samples were allowed to equilibrate ␣ sodium phosphate (pH 7.2), with N -p-tosyl-L-lysine chloromethyl ketone (10 for 2 min at the desired temperature before spectra were recorded. Spectral ␮g/ml) and phenylmethylsulfonyl ﬂuoride (50 ␮g/ml). Cells were then lysed by deconvolution was used to determine the fractions of His-on and His-off sonication, and unbroken cells were removed by ultracentrifugation for 45 species in the observed spectrum as described by Bandarian et al. (9). The min at 40,000 ϫ g. thermodynamics of the conversion of His-off to His-on forms of MetH was Sodium chloride (500 mM)/20 mM sodium phosphate (pH 7.3) was added to determined by initially calculating the equilibrium constant (K ϭ % His-off/% the lysate, and 10–15 ml of the lysate was applied to a HiTrap chelating column His-on) at each temperature, and then using those values to construct a van’t

Datta et al. PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4119 Downloaded by guest on October 1, 2021 Hoff plot. The enthalpy (⌬H) and entropy changes (⌬S) were calculated from solutions were prepared either under native conditions in 0.05 M Tris⅐HCl (pH a linear ﬁt to Eq. 1 8.0)/1 mM EDTA or after denaturation. After reaction with DTNB, the absorbance at 412 nm was measured at 25°C. ln K ϭϪ⌬H/RT ϩ ⌬S/R [1] Mapping the Disulfide Bond by Liquid Chromatography and Mass Spectrometry. The free energy differences were calculated from Eq. 2 The protocol for mapping the position of the disulﬁde bond in samples by LC-MS is included in SI Text. ⌬G ϭ ⌬H Ϫ T⌬S [2] ACKNOWLEDGMENTS. We thank Janet L. Smith (University of Michigan) and Similar experiments were performed with I690C/G743C MetH(649–1227). Catherine Drennan (Massachusetts Institute of Technology, Cambridge, MA) for helpful comments on the manuscript. Diffraction data were collected at Thiol Titration with 5,5؅-Dithio-bis(2-nitrobenzoic Acid) (DTNB). The free thiol GM/CA CAT at the Advanced Photon Source. These studies were supported in content of MetH(649–1227) and I690C/G743C MetH(649–1227) samples was part by National Institutes of Health Grants GM29408 (to R.G.M.) and Ϫ1 Ϫ1 analyzed by reaction with DTNB using an ␧412 of 13600 M cm (33). Enzyme GM16429 (formerly to M.L.L., currently to Janet L. Smith).

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4120 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800329105 Datta et al. Downloaded by guest on October 1, 2021