Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

Ronald J Jenkins,Kyle A Heslip,Jennifer L Meagher,Jeanne A Stuckey,Garry D Dotson

doi:10.1074/jbc.m114.564278

Abstract

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-β-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.

Highlights

Peptide RJPXD33 binds to and inhibits both LpxA and LpxD acyltransferases
The first and third enzymes of the Raetz lipid A pathway (UDP-N-acetylglucosamine acyltransferase, LpxA; UDP-3O-(R-3-hydroxyacyl)-glucosamine acyltransferase, LpxD) are type II acyl-carrier protein (ACP)3-dependent acyltransferases. Both enzymes utilize R-3-hydroxymyristoyl-ACP and UDP-glucosamine core substrates (Fig. 1) in Escherichia coli [8, 9] and display a unique left-handed ␤-helix motif that was first identified in LpxA (10 –12)
The peptide binds to acyltransferases in a mutually exclusive manner with the acyl-ACP substrate

Summary

Background

Peptide RJPXD33 binds to and inhibits both LpxA and LpxD acyltransferases. Results: The crystal structure of the antibacterial peptide RJPXD33 complexed to E. coli LpxA was determined. The first and third enzymes of the Raetz lipid A pathway (UDP-N-acetylglucosamine acyltransferase, LpxA; UDP-3O-(R-3-hydroxyacyl)-glucosamine acyltransferase, LpxD) are type II acyl-carrier protein (ACP)3-dependent acyltransferases. Both enzymes utilize R-3-hydroxymyristoyl-ACP and UDP-glucosamine core substrates (Fig. 1) in Escherichia coli [8, 9] and display a unique left-handed ␤-helix motif that was first identified in LpxA (10 –12). Such structural and functional similarities provide the basis for the possibility that a single inhibitor that targets both enzymes could be identified. Acyltransferase binding studies using truncations of RJPXD33 were undertaken to provide biochemical verification of the structural data obtained

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Data set

Kd LpxD