2025 Theses Doctoral

Structural Insights into Lipid A–Binding Enzymes in the Bacterial Inner Membrane

Zinkle, Allen Peter

Integral membrane enzymes are of particular importance to the cell, as they enable diverse and essential biological processes including the biosynthesis or chemical modification of vital structural components within the membrane. A primary focus of the Mancia laboratory is understanding the key mechanistic processes utilized by these enzymes at the molecular level, often through the structural determination of small (< 100 kDa) membrane proteins with or without their cognate substrates. Here, I will present my investigation of two integral membrane enzymes from Gram-negative bacteria, each acting upon lipid A in the inner membrane.

The O-antigen ligase (WaaL)
In Gram-negative bacteria, a major component of the outer membrane are endotoxins that coat the outer leaflet known as lipopolysaccharide (LPS) molecules. LPS are critically important and provide both structural integrity as well as a protective barrier against antibiotics and other harmful molecules. LPS molecules are comprised of a lipid A anchor, a core oligosaccharide, and a variable O-antigen polysaccharide, which are assembled along either the inner leaflet of the inner membrane or in the periplasm through separate pathways involving multiple different enzymes. The final step in LPS biosynthesis – attachment of O-antigen to the lipid A-core oligosaccharide – is catalyzed by the O-antigen ligase known as WaaL.

At the time of my joining the lab, the structure of WaaL from Cupriavidus metallidurans (CmWaaL) was being determined by a post-doc in the lab, who trained and recruited me on the project to help perform additional cryo-EM screenings and to design, clone, and express mutants in E. coli, based on the CmWaaL structure, for functional investigation. Presented here are my data related to the CmWaaL investigation, as well as data related to my structural investigation into the enzyme from E. coli (EcWaaL). This work provides a structural and functional framework to understand the mechanism underlying the final step of LPS biosynthesis.

The first variant of mobilized colistin resistance (MCR-1)
Multidrug-resistance in bacteria constitutes a rising and urgent public health crisis. In response to this increasing threat, an older class of drugs known as polymyxins – the use of which was halted decades ago primarily due to adverse nephrotoxic side effects – has been used as a last-resort in the clinic to treat multidrug-resistant infections. Polymyxins are small cationic polypeptides that electrostatically target the anionic lipid A component of LPS molecules along the outer membrane, thereby disrupting the membrane and lysing the cell.

One of the most common resistance mechanisms to polymyxins in model organisms such as E. coli and S. enterica involves the chemical modification of lipid A in the inner membrane by a group of integral membrane enzymes known as phosphoethanolamine (PEtN) transferases. Specifically, PEtN transferases, which include the chromosomally encoded EptA and the plasmid-encoded variants of mobilized colistin resistance (MCR-1–10), confer resistance by attaching cationic PEtN from donor phosphatidylethanolamine (PE) substrate to either of the two phosphate groups of lipid A prior to its incorporation into LPS and transport to the outer membrane.

While a structure of EptA has been determined by X-ray crystallography, no molecular-level data exists to validate the binding sites for both donor and acceptor substrates, prohibiting a full understanding of the mechanism underlying PEtN transferase-mediated polymyxin resistance. Here, I present the cryo-EM structure of MCR-1 in complex with both PE and lipid A ligands, revealing two distinct binding sites for the two substrates. I also present biochemical and computational data that suggest PEtN transferases undergo a unique and major conformational change to enable acceptor substrate modification. This mechanism may also be applicable to other phospholipid transferases. We hope this ongoing work will contribute to the understanding of both PEtN transferases in addition to other related enzymes, as well as to the development of novel therapeutics to combat rising rates of multidrug-resistance.

Structural insights into polyisoprenyl-binding glycosyltransferases
Many membrane-bound or-associated glycosyltransferases, including WaaL, depend on polyisoprenyl-phosphate (PP) lipid carriers to carry out their roles in glycoconjugate biosynthesis. Glycosyltransferases that bind PP ligands (PP-GTs) are present across all domains of life and accordingly engage a diverse array of substrates. Presented here is a review I wrote with another graduate student in the lab (R. Morgan) describing the current structural landscape with respect to PP-GTs and potential directions that might be taken to improve our understanding of this diverse enzyme class.