- Home - Introduction - Projects - People - Publications - Vacancies -

The crystal structure of Chlorella virus capping enzyme has been solved by mir methodology and refined at 2.5 Å resolution. The crystals contain two independent molecules in the asymmetric unit, and these two molecules adopt very different conformations, which we refer to as "open" and "closed". These two conformations differ by a rigid body movement of the small domain relative to big domain, closing the gap between them as the protein goes from open to closed conformation. Both molecules contain non-covalently complexed GTP at the active site. Interestingly, the conformations of the triphosphate chain of of the bound GTP are very different for the open and closed form of the capping enzyme

Comparison with DNA ligase demonstrates that the conserved sequence motifs are indeed structurally similar. The main chain atoms of the first four motifs and the first part of the fifth motif superimpose well. The base is buried in a similar pocket and most of the residues that are involved in binding to the ribose and phosphate groups of the ligands are conserved. In contrast, the residues that confer the specificity of GTP over ATP are found outside these motifs. Motif 6 is the only part of domain 2 that appears homologous to to domain 2 of ligase. However, superimposing these residues reveals that several beta strands in domain 2 of the two enzymes align roughly with each other.

Soaking the crystals with excess manganese chloride and GTP results in guanylation of the enzyme to yield the enzyme-GMP adduct, an intermediate in the enzyme catalysed pathway. However, this reaction only takes place in the closed form of the enzyme, showing that domain closure is required for guanylation. The conformation of the triphosphate chain of GTP is suitable for in-line attack only in the closed form of the enzyme. In the open form the only hydrogen bonds between GTP phosphate and enzyme are between the alpha phosphate and residues Lys 82 and Lys 234. In contrast, there are many hydrogen bonds between the entire triphosphate chain and both domain 1 (Lys 82, Arg 106, Arg 228, Lys 234, Lys 236) and domain 2 (Asp 244, Arg 295, Lys 298, Asn 302) in the closed form. This results not only in a different, but also a more ordered and well defined triphosphate chain. In addition, domain closure brings negatively charged residues from both domains together around the manganese binding site. However, the manganese ion makes no close contact with the protein.

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.