Membrane binding mechanism of an RNA virus-mapping enzyme

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the struc ture and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWH LPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present t hat induced a structural change in the peptide from a random coil to a part ially cu-helical conformation. NMR structure shows that the Lu-helix is amp hipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer a ssociation of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.