Structural and Functional Investigation of Bacterial Membrane Biosynthesis

Integral membrane enzymes contribute a unique repertoire to the cell, as they are capable of synthesizing products from substrates of different chemical character at the membrane-water interface. Membrane-embedded enzymes are often responsible for the synthesis of important components of the cellular membrane and contribute to the structural integrity of the cell, maintenance of cellular homeostasis and signal transduction. One of the main focuses of Dr. Filippo Mancia’s laboratory is understanding how enzymes complete these functions by investigating, at an atomic level, the determinants of substrate binding and catalysis within the membrane and at the membrane surface. Here I will present my investigation of two such integral membrane enzyme systems, which are responsible for the synthesis and processing of membrane-embedded molecules in bacteria. Phosphatidylinositol-phosphate Synthase (PIPS) Phosphaitylinositol (PI) is an essential lipid component in mycobacteria, demonstrated by loss of viability when PI is reduced to 50% of wild-type levels. Phosphatidylinositol (PI) is required for the biosynthesis of key components of the cell wall, such as the glycolipids phosphatidylinositol-mannosides, lipomannan and lipoarabinomannan. For these molecules, PI serves as a common lipid anchor to the membrane. In Mycobacterium tuberculosis, the disease causing pathogen of tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response. Therefore, the enzyme responsible for PI synthesis in this organism is a potential target for the development of anti-tuberculosis drugs. The defining step in phosphatidylinositol biosynthesis is catalyzed by a member of the CDP-alcohol phosphotransferase enzyme family. The enzyme uses CDP-diacylglycerol as the donor substrate, and either inositol in eukaryotes or inositol-phosphate in prokaryotes as the acceptor alcohol of the synthesis reaction. In prokaryotes, phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP-alcohol phosphotransferase family) catalyzes this reaction to yield phosphatidylinositol-phosphate, which is then dephosphorylated to PI by an uncharacterized enzyme. Structures of PIPS from Renibacterium salmoninarum (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the location of the acceptor site as well as molecular determinants of substrate specificity and catalysis of the enzyme. However, RsPIPS has low activity relative to PIPS from Mycobacterium tuberculosis (MtPIPS) and the two share only 40% protein sequence identity. Therefore, these initial structures have limited potential for meaningful homology modeling and drug design. Presented here are the structures of PIPS from Mycobacterium kansasii (MkPIPS), which is 86% identical to MtPIPS, in an apo state to 3.1 Å resolution, in a nucleotide-bound state to 3.5 Å resolution, and in a novel ligand-bound state to 2.6 Å resolution. This work provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens. RodA-PBP2 Complex The cell wall of most gram-negative and gram-positive bacteria (excluding atypical bacteria such as members of Mycoplasmataceae) is composed of peptidoglycan, a mesh of repeating carbohydrates (N-acetylmuramic acid, MurNAc, and N-acetylglucosamine, GlcNAc) cross-linked by small peptides. Peptidoglycan is essential for growth, division and viability of the organism. Any disruption of the biosynthesis of peptidoglycan, whether by genetic mutation, inhibition with antibiotics or degradation by lysozyme, results in bacterial cell lysis. Peptidoglycan helps maintain cell shape and serves as an anchor for accessory proteins and other cell wall components. As essential components of the cell wall, enzymes contributing to the peptidoglycan biosynthetic pathway can be exploited as antibiotic targets. After a hydrophilic peptidoglycan precursor (UDP-MurNAc-pentapeptide) is synthesized in the cytosol, it is attached to the lipid carrier undecaprenyl phosphate (UndP). The lipid-linked precursor (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide or Lipid I) is modified further to undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (Lipid II) by addition of a GlcNAc moiety. Lipid II is then flipped across the membrane to the periplasm where its sugars are polymerized to form the glycan strands of the peptidoglycan mesh. SEDS proteins, essential for maintaining bacterial processes that determine shape, elongation, cell division and sporulation, are integral membrane enzyme that have been implicated in this process as either Lipid II flippases, glycosyltransferases responsible for sugar polymerization, or both. SEDS proteins are also known to form a functional complex with type b penicillin-binding proteins (PBPs), which are known as transpeptidase enzymes, responsible for the crosslinking of peptides in the formation of the peptidoglycan mesh. Though structures of both RodA (a SEDS protein involved in bacterial growth and elongation) and type b PBPs are available, the interaction between the two proteins and their joint enzymatic activity is poorly characterized. Here, I present the preliminary structural characterization of a RodA-PBP2 protein complex by single-particle cryo-electron microscopy (cryo-EM). We hope this ongoing work will contribute to the understanding of these enzymes and to the development of antibiotics to combat antibiotic resistance

Structure-based analysis of CysZ-mediated cellular uptake of sulfate

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils.

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces

Structural basis of lipopolysaccharide maturation by the WaaL O-antigen ligase

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases