Insight into Elongation Stages of Peptidoglycan Processing in Bacterial Cytoplasmic Membranes

Seonghoon Kim,Wonpil Im,Marcos M. Pires

doi:10.1038/s41598-018-36075-y

Abstract

Peptidoglycan (PG) biosynthesis and assembly are needed for bacterial cell wall formation. Lipid II is the precursor in the PG biosynthetic pathway and carries a nascent PG unit that is processed by glycosyltransferases. Despite its immense therapeutic value as a target of several classes of antibiotics, the conformational ensemble of lipid II in bacterial membranes and its interactions with membrane-anchored enzymes remain elusive. In this work, lipid II and its elongated forms (lipid VI and lipid XII) were modeled and simulated in bilayers of POPE (palmitoyl-oleoyl-phosphatidyl-ethanolamine) and POPG (palmitoyl-oleoyl-phosphatidyl-glycerol) that mimic the prototypical composition of Gram-negative cytoplasmic membranes. In addition, penicillin-binding protein 1b (PBP1b) from Escherichia coli was modeled and simulated in the presence of a nascent PG to investigate their interactions. Trajectory analysis reveals that as the glycan chain grows, the non-reducing end of the nascent PG displays much greater fluctuation along the membrane normal and minimally interacts with the membrane surface. In addition, dihedral angles within the pyrophosphate moiety are determined by the length of the PG moiety and its surrounding environment. When a nascent PG is bound to PBP1b, the stem peptide remains in close contact with PBP1b by structural rearrangement of the glycan chain. Most importantly, the number of nascent PG units required to reach the transpeptidase domain are determined to be 7 or 8. Our findings complement experimental results to further understand how the structure of nascent PG can dictate the assembly of the PG scaffold.

Highlights

Bacterial cell wall biogenesis is critically important to the viability of bacteria and underpin bacterial pathogenesis and human microbiome interactions[1]
How do elongated lipid-anchored PG intermediates behave within bacterial membranes? How does the size of the nascent PG control association with PBPs? Most critically, how many elongation steps are needed for the nascent PG to reach the TPase domain? Towards answering some aspects of these fundamental questions, we first modeled and simulated Gram-negative lipid-anchored PG precursors such as lipid II, lipid VI, and lipid XII in membranes that are representative of Gram-negative bacteria (Figs 1 and 2)
We have investigated the structure and dynamics of isolated lipid II, its elongated forms, and the penicillin-binding protein 1b (PBP1b)-nascent PG complex in Gram-negative bacterial-mimetic membranes to understand how nascent PG structures control PG elongation and processing

Summary

Introduction

Bacterial cell wall biogenesis is critically important to the viability of bacteria and underpin bacterial pathogenesis and human microbiome interactions[1]. Given the importance of GTase activity for PG biosynthesis, it is important to understand how glycan chain elongation can control substrate access and priming Towards these goals, the crystal structure of GTase from Staphylococcus aureus was previously solved with the antibiotic moenomycin to gain structural insight into the mechanism of action of moenomycin[13]. Transmembrane helix subdomain was recently published[14] Based on these works, we have set out to model nascent PG in complex with PBP1b with the ultimate goal of gaining structural insight into PBP-PG interactions. It was established that during glycan elongation by GTase, glycan chains are stitched together unidirectionally before being loaded onto the existing PG scaffold These studies did not provide atomistic details about the dynamics of lipid-bound PG precursors in a membrane environment and within PBPs. How do elongated lipid-anchored PG intermediates behave within bacterial membranes? E. coli PBP1b was modeled and simulated in the presence of nascent PG (PBP1b-lipidXX complex) to gain atomistic insight into the dynamics of nascent PG in association with PBP1b (Fig. 1)

Methods

Results

Conclusion