Reconstitution of the LnsAB lipoprotein N-acylation system complex from Staphylococcus aureus and characterization of substrate preference.

In Staphylococcus aureus, over 50 different types of lipoproteins are tethered to the membrane surface through acylation of an N-terminal cysteine residue where they perform multiple cellular roles, from nutrient transport to displaying virulence factors. Lipoproteins are also key focal points for detection by the toll-like receptor 2 family of innate immunity. While all staphylococci initiate lipoprotein biosynthesis by attaching a thioether-linked diacyl glycerol moiety, subsequent α-amino tailoring occurs in a species dependent manner that attenuates toll-like receptor 2 signaling. A two-gene system encoding an NlpC/P60 superfamily enzyme (LnsA) and a CAAX protease/bacteriocin-processing family integral membrane protein (LnsB) is required for lipoprotein N-acylation in S. aureus, but little is known regarding the mechanism. Herein, we show that the LnsA N-terminal α-helix is a canonical cleaved signal peptide that is unnecessary for membrane retention. We use protein complex modeling with diacylated lipopeptide substrate and substituted cysteine crosslinking to demonstrate a key loop on LnsA interacts with LnsB in a multimeric complex. Using targeted site mutagenesis, a Cys-His catalytic dyad common to NlpC/P60 superfamily members is defined while no CAAX protease/bacteriocin-processing motif residues were essential. Reconstitution using recombinant LnsA and LnsB with lipopeptide substrate confirmed that both proteins are required for catalysis in vitro, and that the SN