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.

Structure of the þ domain. The crystal structure of CR-VI þ was solved to 2.2 Å resolution in space group P2 1 2 1 2 1 with two molecules, disulphide-linked at residue C 1877 , in the asymmetric unit, assuming a 'head-to-toe' conformation ( Supplementary Fig. 1 ). There are no significant differences between the molecules, which have a slightly twisted, bi-lobed shape, composed of two globular domains, the larger corresponding to CR-VI (residues B1,616-1,883) and the smaller to the þ domain (1,884-2,005; Fig. 1b ). Although composed of only B120 residues in Pneumovirinae, the size of the þ domain varies greatly within the Mononegavirales order, reaching B240 residues in Rhabdoviridae. In hMPV, it consists of six a-helices (a þ 1-6). Helix a þ 6 contains the K-K-G motif ( Supplementary Fig. 2 ) and together with a þ 1 leans over the active cleft of CR-VI. Helix a þ 4, and to a lesser extent helices a þ 1, a þ 5 and a þ 6, packs down on l 1650-1666 , the second half of a long loop (residues 1,635-1,666) that swerves around the CR-VI domain ( Fig. 1b and Supplementary Fig. 3 ; 'l' is used throughout the paper to denote loops). The l 1650-1666 region, which contains a small helix (a-a), acts as a fulcrum allowing the þ domain to pivot relative to the CR-VI domain. Helix a þ 3 varies in length, from 4½ turns (in most crystals) to 6 (in Protein Data Bank (PDB) 4UCY), and the loop between a þ 2 and a þ 3 is always disordered ( Supplementary Fig. 3 ). In crystals of the monomeric C 1877 A mutant (space group P3 1 Fig. 2 ), using GpppGGGACAAGU substrates that were methylated beforehand at N7 of G or 2 0 O of N1, to specifically monitor 2 0 O or N7-MTase activities, respectively. Mutants are listed against a yellow, green or red background, to indicate that the altered residue belongs to l 1650-1666 , the rest of the CR-VI domain, or the þ domain, respectively. They are also grouped according to whether they change the K-D-K-E tetrad, SAM P, SUB P or NS P. The results highlight the importance of essential SUB P residues (such as the K-K-G lysines K 1991 , K 1992 and K 1995 and l 1650-1666 residues H 1659 and R 1662 ) for both 2 0 O-and N7-methylation. All tetrad residues are crucial for region is further enlarged, as helix a þ 2 completely unfolds and a þ 3 unwinds to 3½ turns. 12 , such as the long N-terminal loop and the position of helix aX. In its active core, however, it better resembles RrmJ-type flavivirus MTases (for example, 3EVF 9 ; Supplementary Fig. 4 ). Most notably, hMPV and flaviviruses share an unusually long (B10 residues), flexible b2 l (that is, the loop immediately following the b2-strand), which forms the SAM-binding pocket ( SAM P) along with loops b1 l and b4 l, shielding it from the solvent (Fig. 1b, c) .