DUF916 and DUF3324 in the WxL protein cluster bind to WxL and link bacterial and host surfaces.

Bacterial WxL proteins contain peptidoglycan-binding WxL domains, which have a dual Trp-x-Leu motif and are involved in virulence. It was recently shown that WxL proteins occur in gene clusters, containing typically a small WxL protein (which in the mature protein consists only of a WxL domain), a large WxL protein (which contains a C-terminal WxL domain with N-terminal host-binding domains), and a conserved protein annotated as a Domain of Unknown Function (DUF). Here we analyze this DUF and show that it contains two tandem domains-DUF916 and DUF3324-which both have an IgG-like fold and together form a single functional unit, connected to a C-terminal transmembrane helix. DUF3324 is a stable domain, while DUF916 is less stable and is likely to require a stabilizing interaction with WxL. The protein is suggested to have an important role to bind and stabilize WxL on the peptidoglycan surface, via the DUF916 domain, and to bind to host cells via the DUF3324 domain. AlphaFold2 predicts that a β-hairpin strand from DUF916 inserts into WxL adjacent to its N-terminus. We therefore propose to rename the DUF916-DUF3324 pair as WxL Interacting Protein (WxLIP), with DUF916, DUF3324 and the transmembrane helix forming the first, second and third domains of WxLIP, which we characterize as peptidoglycan binding domain (PGBD), host binding domain (HBD), and transmembrane helix (TMH) respectively.