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.

In Wesselsbron (flavi-)virus, b2 l is found in closed or open conformations, either packing up against SAM (PDB: 3ELW) or exposing it to the solvent (3EMB) 13 , changes that may assist SAM uptake and/or SAH expulsion. In CR-VI þ , b2 l similarly assumes alternate conformations (Fig. 4c,d) . In the 'closed' form, the ligand's ribose group is hydrogen bonded to D 1725 , and-via a water-to D 1722. Loops b1 l and b4 l also show a degree of flexibility. b1 l residue E 1697 , conserved in paramyxo-and filoviruses, forms a hydrogen bond with the NH 2 group of SAM and is essential for both MTase activities (Fig. 3b) . However, in the absence of SAM, its side chain either moves into the sub-pocket that normally accommodates the NH 2 group, or, more markedly, turns towards the solvent in the direction of b2 l, which in this case assumes the 'open' position ( Fig. 4d) . b4 l forms a side wall of SAM P, and together with aD and b5 also defines a deep, hydrophobic cavity not present in other MTases, termed NS P (or nucleoside-binding pocket). Although a role for NS P has yet to be determined, the pocket binds the adenosine moiety of SAM or ATP, soaked at high (25 mM) concentrations into CR-VI þ crystals, causing b4 l to impinge onto SAM P (Fig. 4c,d; when SAM is used for soaking, one SAM molecule occupies SAM P, and another binds to NS P). GTP was not observed in NS P, but this may reflect a lower soaking concentration (2.5 mM), due to GTP's poor solubility. With the exception of E 1781 , the amino acids lining NS P are poorly conserved beyond the Pneumovirinae, and mutating key NS P residues barely affects the MTase activities (Fig. 3b) . CR-VI, finally, contains a Zn-finger, which is not conserved beyond the Pneumovirinae subfamily and links the small a-d 0 to the rest of the structure (Fig. 1b) . During the preparation of this manuscript, a 3.8-Å structure of VSV L, obtained by electron cryo-microscopy, was published 14 . The CR-VI (or MTase) part of VSV resembles that of hMPV, but apparently lacks a deep NS P pocket (PDB: 5a22, Fig. 5 ). The þ domain (C-terminal domain) is more elaborate in VSV than in hMPV, containing extra regions N and C terminal of helix a þ 6. The helix itself, however, appears well conserved, both in length and in position. Although a K-K-G motif is not present in VSV, it does contain an arginine (R 2038 ) strictly conserved among the Rhabdoviridae, which is structurally equivalent to hMPV's K 1995 , the second lysine of K-K-G motif.