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

In Staphylococcus aureus, over fifty 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 the display of virulence factors. Lipoproteins are also key focal points for detection by the Toll-like receptor 2 (TLR2) 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 TLR2 signaling. A two-gene system encoding an NlpC/P60 superfamily enzyme (LnsA) and a CAAX protease and bacteriocin-processing (CPBP) 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 cysteine crosslinking to provide evidence that 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 CPBP motif residues were essential. Reconstitution using recombinant LnsA and LnsB with lipopeptide substrate confirmed that both proteins are required for catalytic activity in vitro, and that the SN1 position of phosphatidylglycerol is the preferred acyl chain substrate donor. This work begins to define the LnsAB complex, and further underscores the rich source of unique acylation biochemistry that has evolved in bacterial lipoprotein N-terminal modification pathways.