As described recently in Nature (Lake et al., in the press), Schindelin and colleagues have solved X-ray crystal structures of MoaD in complex with MoeB, representing the first structures of a ubiquitin-like protein bound to its activating enzyme. Three structures — of the apo-complex, the ATP-bound form and the adenylated MoaD form (shown here) — provide an evolving picture of the adenylation reaction. The structures reveal a MoaD2–MoeB2 tetramer, in which both of the MoeB subunits (dark and light gray) contribute to each active site. Several active site residues are strictly conserved between MoeB and E1 (these are shown in green for one monomer and yellow for the other). Based on their structures, Lake et al. propose a reaction mechanism for the adenylation of MoaD, and by analogy, ubiquitin.
In the proposed mechanism, a carboxylate oxygen of the C-terminal Gly 81 of MoaD (the adenylated MoaD is shown in an all-bonds representation) directly attacks the α-phosphate of the ATP molecule. The repulsion between these two negatively charged species is presumably mitigated by a nearby Mg2+ ion, predicted to be coordinated by Asp 130 of MoeB (green, to the left of MoaD). Consistent with this, the authors show that mutation of Asp 130 severely reduces the activity of the enzyme. Binding of ATP appears to induce a kink at the α-phosphate, which promotes cleavage of the bond between the α- and β-phosphates. Several conserved residues in MoeB, including Arg 14 (yellow) and Arg 73 (green residue behind Arg 14) are proposed to stabilize the negative charge on the β-phosphate of the leaving group; the authors show through mutagenesis that at least one positive charge in this region is required for activity.
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