X-ray structure and activities of an essential Mononegavirales L-protein domain

Paesen, Guido C.; Collet, Axelle; Sallamand, Corinne; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Grimes, Jonathan M.

(1) (9) (2) , which runs somewhat differently in VSV. The l 1650-1666 peptide on which the þ domain rests (yellow) also has a homologue in VSV. The Zn-finger, however, is not conserved, and a-helices B and Z are not fragmented. Helix aE, an element of the standard MTase topology (Fig. 1c) , is present in VSV (3), as a result of which NS P may have disappeared. aX is at a different location (4), and is preceded by an extra helix (ax 0 (5)). E 1833 , expected to belong to the K-D-K-E tetrad from sequence alignments, is buried in the structure and does not reach the surface of the catalytic pocket (6) , and the position normally taken by the K-D-K-E glutamate is occupied by T 1831 . The þ domain of VSV is tilted, compared with that of hMPV, and more elaborate. Helices a þ 1, a þ 2, a þ 3, a þ 5 and a þ 6 are conserved, but the a þ 1-a þ 2 loop is replaced by an extra helix (a þ 1 0 (7)). a þ 4 is absent, whereas a þ 5 is enlarged and immediately follows a þ 3 (8). The 2-residue loop connecting a þ 5 and a þ 6 in hMPV is replaced by a 34-residue coil carrying a small three-stranded b-sheet (9) . Helix a þ 6 seems best conserved between the two þ domains, although a K-K-G motif is not present in VSV. However, R 2038 , which is strictly conserved in Rhabdoviridae, takes the place of K 1995 ((10) and alignment below). In VSV, the þ domain is extended beyond a þ 6 with a 65-residue, partly helical, but mainly unstructured polypeptide (in grey (11)). Colour scheme and labelling are as in Fig. 1b. (b) . Alignment of a þ 6-helices from Mononegavirales L proteins. K-K-G motif residues are highlighted in red; the arginine replacing the second lysine of the motif in Filoviridae and Rhabdoviridae is highlighted in magenta. Red letters indicate other (less strictly) conserved residues, except for the G that replaces the first lysine of the K-K-G motif in most Rhabdoviridae (blue). particular, the site adjoining SAM P, which holds the nucleotides undergoing methylation, has become a more elaborate, but narrower and possibly therefore, higher-affinity substrate-binding pocket (termed SUB P as it accommodates the nucleotide undergoing methylation). Consistently, electron density is found in SUB P following soaking or co-crystallization with GTP, whereas in other MTases added GTP predominantly shows up in the capbinding pocket. In particular, helix a þ 6 and the þ domainaffiliated l 1650-1666 help shape SUB P through the side chains of K 1991 and K 1995 (of the K-K-G motif), and of H 1659 and R 1662 , respectively (Fig. 4b) . The marked decrease in MTase activity of mutants altered at these residues (Fig. 3b) illustrates the importance of SUB P in correctly presenting the substrate nucleotides to SAM. Nevertheless, the pocket is too spacious for a single nucleotide, and the electron density in SUB P from a number of soaked crystals suggests that bound GTP often assumes more than one orientation. In structures where GTP could be fitted with confidence, the guanosine moiety predominantly interacts with l 1650-1666 residues H 1659 and R 1662 , and with K 1991 and (K-D-K-E residue) K 1673 , which clamp the guanine (Fig. 4b) . Unusually for cap-MTases, K 1673 is not part of aZ, but instead resides on the small z 0 (3 10 )-helix (Fig. 1b,c) . Whether any of the observed positions of GTP reflects in vivo binding of N 1 (as part of a transcript) is unclear; in MTase-RNA complexes (PDB: 1AV6 (ref. 17) , 4N49 (ref. 16) ), N 1 is situated much closer to the K-D-K-E t