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. 2006 Dec 26;45(51):15188-96.
doi: 10.1021/bi061396f.

Structure of ATP-bound human ATP:cobalamin adenosyltransferase

Heidi L Schubert  1 , Christopher P Hill
Affiliations

Structure of ATP-bound human ATP:cobalamin adenosyltransferase

Heidi L Schubert et al. Biochemistry. .

Abstract

Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B12) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 A crystal structure of ATP bound to hATR refined to an Rfree value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

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Figures

Figure 1
Figure 1
Schematic outline of the reaction catalyzed by ATP:cobalamin adenosyltransferase.
Figure 2
Figure 2
Structure of hATR. A) Side view of the hATR monomer. The trace is colored blue from the N-terminus to red at the C-terminus. The N-terminal portion is only ordered in the presence of bound ATP; molecule C is shown. B) Top view of the hATR trimer. Molecules A, B and C are colored yellow, blue and green, respectively. ATP molecules (magenta) with two Mg2+ ions each (red) are bound at the AC and CB interfaces underneath the ordered N-terminal β-strands. The apo-active site sits at the BA interface. The trimer interface formed by the supercoil of helices α1 and α4 contains alternating Glu/Arg residues (Glu84/Arg195 and Glu91/Arg191 - cyan). This charged network connects via Glu84 to the ATP binding site though a hydrogen bond to invariant Arg194 (magenta). A chloride ion has been modeled in the center of the charged ring (orange). Two rings of phenylalanine side chains lie on the trimer interface below the Arg/Glu pairs and are omitted from this figure for clarity. C) Sequence of the hATR with invariant resides highlighted in red, as determined by the extended sequence alignment included as supplemental data. Secondary structure is shown above and colored as in Figure 2a. Colored triangles below the sequence denote the functional role of the invariant residues (Table 2), ATP-binding – red, cobalamin binding pocket – blue, structural function – cyan. Figure 2a,b, Figure 3, Figure 4 and Figure 5 were made with PYMOL (45).
Figure 3
Figure 3
Side view of the hATR active sites. A) Surface of the apo-active site (at the BA interface) reveals several exposed invariant residues. The figure is colored as in figure 2b with invariant residues in darker hue. The right-hand molecule, molecule A (yellow), is only ordered from residue 79. B) An ATP-bound active site (the AC active site is shown in the same orientation as in panel A) reveals the ordered N-terminal residues of the right-hand molecule, molecule C (green), starting at residue 57, and wrapping over the top of the bound ATP (ball-and-stick). C) All three monomers are aligned revealing close structural similarity except at the N-terminus where binding of ATP causes ordering of ~22 N-terminal residues in molecules B and C. D) Detailed view of the apo active site (the BA interface). E) The ATP-bound active site at the AC interface with residues involved in binding ATP or positioned in the putative cobalamin binding pocket (Arg186, Phe170, Phe221, Asp90) labeled. All labeled residues are invariant except Ser68 and Ser69. F) The ATP-bound active site of the BC interface reveals slight differences in the extreme N-terminus of molecules B and C.
Figure 4
Figure 4
ATP binding site. A) The ATP binding site is shown in stereo. Invariant residues that are involved in forming hydrogen bonds to the bound ATP are shown explicitly. The hydrogen bond from non-conserved Ser69 extends from its main chain carbonyl not its hydroxyl side chain. Two Mg2+ ions interact with the tri-phosphate moiety. Arg194 recognizes the adenosine and connects this interaction to the trimer core. B) Schematic representation of ATP binding site and interactions with hATR residues. Hydrogen bonds are shown as dashed lines with distances shown in Ångstroms. Waters and Mg2+ are shown as closed and open circles. The secondary Mg which is likely not physiologically relevant is shown in grey. Ser68, Phe83 and Gly87 are conserved but not invariant.
Figure 5
Figure 5
Model of base-off cobalamin bound to the active site, shown in stereo in the same orientation as figure 3e. The model is not intended to imply specific residue interactions, but rather to provide a sense of scale and demonstrate the plausibility of the apparent cobalamin binding pocket. The corrin ring (blue) is positioned ~4Å away from the ATP (cyan) and the amide side chains of the A- and B-rings point towards the base of the binding cleft. The benzimidazole tail (white) is extended away from the structure in a position that is highly speculative in the absence of additional structural information.
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