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.

Cap methylation is catalysed by S-adenosylmethionine (SAM)dependent methyltransferases (MTases), which position a SAM molecule next to the target atom on the RNA, enabling the direct transfer of a methyl group and converting SAM into S-adenosylhomocysteine (SAH). In 2 0 O-MTases, a conserved K-D-K-E tetrad potentiates methyl transfer 2 . As yet, the boundaries of CR-VI, the putative MTase of L, are not well (a) Domain organization of hMPV L, with at its C terminus the 46.5 kDa CR-VI þ domain (residues 1,599-2,005), comprising CR-VI (green), which contains the K-D-K-E motif typical for 2 0 O-MTases, and the þ domain (red), carrying the K-K-G motif (blue). Boundaries of CR-I to -V are approximate. CR-III contains the G-D-N-Q signature motif for polymerase (RdRP) activity, and CR-V contains the HR motif for PRNTase activity. (b) Cartoon representation of the CR-VI þ crystal structure, from amino (N) to carboxy (C) terminus (no structure could be assigned to the first B18 residues). The þ domain is shown in red, with K 1991 and K 1995 of the K-K-G motif in stick format. The CR-VI (MTase) domain is coloured purple (b-strands) and green (helices and loops), except for b1 l, b2 l and b4 l (the loop regions C terminal of b-strands 1, 2 and 4 that form SAM P; orange), and l 1650-1666 (which disengages itself from the main CR-VI-fold to interact with the þ domain; yellow). Nomenclature of helices and strands follows that used for other MTases (c). The pale-blue sticks show the K-D-K-E motif. A Zn-ion (silver sphere) is co-ordinated by H 1766 , H 1798 , C 1802 and C 1805 . (c) Schematic representation of the secondary structure of a prototypical SAM-dependent MTase (top) and of the hMPV CR-VI domain (bottom). Helices are in green, strands in light purple and coils in blue, except for b1 l, b2 l and b4 l (orange) and l 1650-1666 (yellow). CR-VI displays some deviations from the prototypical SAM-MTase fold, some of which it shares with other RNA-MTases, including the long N-terminal coil, a longer aD and an extra helix (aX) at the C terminus. aE is absent, whereas, atypical for viral MTases, aB is fully formed. CR-VI, moreover, has an unusual b-sheet; it lacks b3, but this is compensated for by the addition, at the other end of the sheet, of a new strand (b0), which glues the start of the N-terminal coil to the main structure. Also unusual is the fragmentation of aZ (resulting in the small z 0 -helix).