Abstract
Scribble is a highly conserved regulator of cell polarity, a process that enables the generation of asymmetry at the cellular and tissue level in higher organisms. Scribble acts in concert with Disc-large (Dlg) and Lethal-2-giant larvae (Lgl) to form the Scribble polarity complex, and its functional dysregulation is associated with poor prognosis during viral infections. Viruses have been shown to interfere with Scribble by targeting Scribble PDZ domains to subvert the network of interactions that enable normal control of cell polarity via Scribble, as well as the localisation of the Scribble module within the cell. The influenza A virus NS1 protein was shown to bind to human Scribble (SCRIB) via its C-terminal PDZ binding motif (PBM). It was reported that the PBM sequence ESEV is a virulence determinant for influenza A virus H5N1 whilst other sequences, such as ESKV, KSEV and RSKV, demonstrated no affinity towards Scribble. We now show, using isothermal titration calorimetry (ITC), that ESKV and KSEV bind to SCRIB PDZ domains and that ESEV unexpectedly displayed an affinity towards all four PDZs and not just a selected few. We then define the structural basis for the interactions of SCRIB PDZ1 domain with ESEV and ESKV PBM motifs, as well as SCRIB PDZ3 with the ESKV PBM motif. These findings will serve as a platform for understanding the role of Scribble PDZ domains and their interactions with different NS1 PBMs and the mechanisms that mediate cell polarity within the context of the pathogenesis of influenza A virus.
Highlights
Cell polarity refers to the differential distribution of macromolecules within a cell that allows it to orientate itself in a specific direction, which is an essential property for correct tissue development, organisation and function [1]
The synthetic codon optimised cDNAs encoding for the human Scribble (SCRIB) PDZ1 domain and SCRIB PDZ2 domain were cloned into the pGex-6P3 vector (Bioneer, Daejeon, Korea) that contained a Glutathione S-transferase (GST)-tag encoding sequence at the N-terminus followed by a human rhinovirus (HRV) 3C protease cleavable linker, as described previously [28]. cDNAs of SCRIB PDZ3 and SCRIB PDZ4 were cloned into the pGil vector (Bioneer) which contained a Hexahistidine-Maltose binding protein (His6-MBP) tag at the N-terminus followed by a tobacco etch virus (TEV) protease cleavable linker described previously [28]
These interactions contrast with previously observed interactions from GST pull-down studies that suggested the ESEV PDZ binding motif (PBM) sequence in the context of recombinant full-length non-structural protein 1 (NS1) was only able to bind to a tandem of PDZ1 and PDZ2 [22]
Summary
Cell polarity refers to the differential distribution of macromolecules within a cell that allows it to orientate itself in a specific direction, which is an essential property for correct tissue development, organisation and function [1]. Examples of cell polarity include the axonal-dendritic directionality of neurons, the migration and asymmetric division of mesenchymal cells, and the apical-basal orientation or planar polarity of epithelial cells [2–6]. For apical-basal, migrating and asymmetrical dividing polarity cell types three key protein complexes are involved in an antagonistic relationship to control cell polarity: the PAR module, the Crumbs module and the Scribble module [8]. These polarity regulatory modules play important roles in determining cell architecture and are highly conserved in higher organisms
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