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The enzyme was cocrystallized with G[5']ppp[5']G (cap analog). This ligand can be regarded as a capped single residue RNA molecule. The crystal form obtained was different to the GTP complex described above and there was only one molecule in the asymmetric unit. The enzyme conformation is similar to that of the open GTP complex, with a sizeable gap between the two domains. All contacts between enzyme and ligand are mediated by domain 1; there are no hydrogen bonds or other close contacts between the ligand and the small domain. One of the guanine groups is bound in the same pocket as GTP. The rest of the ligand molecule turns back with the second purine group stacked against the hydrophobic Ile 86. This residue is part of motif 1 (see section B), and is conserved as a leucine or isoleucine in capping enzymes, ATP dependent ligases and NAD dependent ligases.

The geometry around the alpha phosphate is similar to that of the closed GTP complex, i.e. suitable for inline attack by the active site lysine 82. This is remarkable, since the second reaction (transfer of GMP from enzyme to RNA) is analogous, albeit reversed, to the first (transfer of GMP from pyrophosphate to enzyme). In the first reaction, this reactive alpha phosphate conformation is achieved by domain closure and interactions between the gamma phosphate and motif 6 of the small domain. The second transfer occurs with an open enzyme conformation where the alpha phosphate geometry is largely determined by its covalent link to the 5' end of the mRNA which is bound non-covalenty to domain 1.

The cap analogue G[5']ppp[5']G (blue) bound to the active site of the capping enzyme (red). Motif 1 (green) is displayed with sidechains.

Comparison of the ligand alpha phosphate conformation in the different capping enzyme structures. The geometry is suitable for in-line attack by the active site lysine in the closed GTP complex and in the GpppG complex, but not in the open GTP complex.

A) Capping chemistry
B) Sequence and conserved motifs
C) Structure of mRNA capping enzyme
D) Binding of cap analogue
E) Reaction mechanism
F) Authors and references

These capping enzyme homepages were constructed by Kjell.