Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein-protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport.
Methylation is a fundamental mechanism used in Nature to modify the structure and function of biomolecules, including proteins, DNA, RNA, and metabolites. Methyl groups are predominantly installed into biomolecules by a large and diverse class of S-adenosyl methionine (SAM)-dependent methyltransferases (MTs), of which there are approximately 200 known or putative members in the human proteome. Deregulated MT activity contributes to numerous diseases, including cancer, and several MT inhibitors are in clinical development. Nonetheless, a large fraction of the human MT family remains poorly characterized, underscoring the need for new technologies to characterize MTs and their inhibitors in native biological systems. Here, we describe a suite of S-adenosyl homocysteine (SAH) photoreactive probes and their application in chemical proteomic experiments to profile and enrich a large number of MTs (>50) from human cancer cell lysates with remarkable specificity over other classes of proteins. We further demonstrate that the SAH probes can enrich MT-associated proteins and be used to screen for and assess the selectivity of MT inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide N-methyltransferase (NNMT), an enzyme implicated in cancer and metabolic disorders. The chemical proteomics probes and methods for their utilization reported herein should prove of value for the functional characterization of MTs, MT complexes, and MT inhibitors in mammalian biology and disease.