Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N -acyltransferase

Abstract

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N -acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo–electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt’s substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

7.10 MAG. A Novel Host Monoacylglyceride for In Meso (Lipid Cubic Phase) Crystallization of Membrane Proteins

“…aeruginosa. , The second is the lipoprotein N -acyltransferase, Lnt, from E. coli. , Lnt has half its 57 kDa mass as an α-helical bundle residing in the cytoplasmic membrane, on top of which sits a domain that is a mix of β-strands and α-helices in the form of an αββα sandwich. Finally, the predominantly α-helical adenosine receptor, A2aR, which requires the addition of cholesterol to the mesophase for growth of diffraction quality crystals, was tested.…”

“…7 The crystal structures of AlgE and Lnt obtained using 7.10 MAG are, for the most part, superimposable on those generated using 9.9 MAG. 39,48,60,65 It is very likely that the bilayer thickness and fluidity characteristics of the hosting mesophase formed in these different MAGs and used to grow crystals are slightly different. However, these differences do not show up in the corresponding crystal structures, suggesting that the final form adopted in the crystal reflects the state of the protein assumed in the native membrane which is unlikely to change dramatically in the course of the protein's activity cycle.…”

“…These reside in the extracellular loop that coordinates with the phosphate in the glycerophospholipid substrate and product and that is known to be flexible. 65 Eight structured lipids were modeled into density as 7.10 MAG molecules. Six decorate the outer surface of the transmembrane helical core of Lnt, roughly in a bilayer arrangement as observed with AlgE.…”

Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics

“…18,19 By leveraging signal intensities as a measure of the relative abundance of species in solution, native MS reports on protein−ligand binding affinity, 20−23 and enzyme catalysis. 24,25 We aimed to develop a combination of real-time enzyme activity monitoring and in situ inhibition to yield a mechanistic insight into the MoA of antibiotics. To develop our approach, we selected two Escherichia coli membrane phosphatases UppP and PgpB as model membrane enzymes, having different substrate specificities.…”

“…Omics approaches (genomics, transcriptomics, proteomics, and metabolomics) − can probe phenotypic changes in the cell in response to antibiotic stimuli. In addition to these methodologies, native mass spectrometry (native MS) can capture noncovalent interactions between proteins and substrates, lipids and drugs, presenting a new platform for probing the MoA of antibiotics. , By leveraging signal intensities as a measure of the relative abundance of species in solution, native MS reports on protein–ligand binding affinity, − and enzyme catalysis. , …”