Research Projects

Rationale of the research: Membranes play a crucial role in exchange of information and form a protective barrier to the cell. Along with cell wall in some organisms, membranes are important for survival of the cell and are also targets for small molecules. In higher eukaryotes, Cationic antimicrobial peptides (CAMPs) are vital components of innate immune system that have recently shown to be produced by proteasomal degradation (Goldberg et al, 2025). These CAMPs act on the bacterial membrane to combat infection. Bacteria have evolved ways to overcome CAMPs and, one of the mechanisms is mediated by Multiple Peptide Resistance Factor (MprF), a bi-functional membrane enzyme found in variety of infectious microbes. This dual enzyme has a synthase domain-binds aminoacylated tRNA and transfers amino acid to head group of a lipid molecule (Steinbuch et al,2016). Subsequently, the modified lipid is flipped from inner to outer leaflet, thereby neutralising/introducing a positive charge on the membrane. Thus, MprF modulate property of the bacterial membrane having roles beyond conferring resistance to CAMPs. Although, these enzymes were discovered couple of decades ago, the underlying mechanism governing the varied substrate specificity of MprF from different species and mechanism of substrate binding has remained elusive (Ernst & Peschel, 2011). The structural characterisation of isolated synthase domain and FL enzyme has recently been characterised showing a dimeric architecture along with a monomer for the same enzyme. It is clear from these studies that the enzymes has evolved in organisms to suit their environmental requirements and understanding these interesting enzymes from different organisms is a long term goal. In this proposal, I focus on one model enzyme to understand mechanism of lipid modification and flipping. Objectives: I will use MprF from Pseudomonas aeruginosa to understand mode of substrate binding, origin of substrate specificity, and mechanism of flipping. Hypothesis to be tested: I hypothesise that substrate specificity of MprF is mediated via coordinated structural changes within its synthase and flippase domains. These conformational changes may play a role in conferring resistance to CAMPs. Using Cryo-EM, I aim to capture substrate-bound states of MprF, which will reveal the dynamic transitions that underlie substrate recognition, binding, and membrane interaction. These structural insights will help elucidate a general mechanism of MprF function across bacterial species. Significance to the field of research: The structures of these enzymes (and mutants) with substrates/substrate mimics in different conformational states will provide snapshots, which will help in identification of substrate binding site and further computational design of new molecules. Beyond revealing mechanism of MprF it will also help in structure-guided inhibitor development, offering new avenues to combat antimicrobial resistance in pathogenic bacteria.