The Relative Binding Position of Nck and Grb2 Adaptors Dramatically Impacts Actin-Based Motility of Vaccinia Virus

Abstract

Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to nucleate complex signalling networks. The concept of phase separation has recently changed our appreciation of such multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now explored this parameter in the assembly and operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3-mediated actin polymerization. We generated synthetic networks on Vaccinia by manipulating pTyr motifs in A36 and the unrelated p14 from Orthoreovirus. In contrast to predictions, we find that only specific spatial arrangements of Grb2 and Nck binding sites result in robust N-WASP recruitment, Arp2/3 driven actin polymerization and viral spread. Our results suggest that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are usually conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those that undergo phase transitions.