Biology subjects

Denic, M.

Publications and source records attributed to Denic, M..

3 recordsLinked to original sources

bioRxiv2026

A short C-terminal plug motif in a conserved type 4 pilus component controls pilus-tip localisation and filament biogenesis

Type 4a pili (T4aP), the most widespread and functionally versatile subtype of the type 4 filaments (T4F) superfamily, are functionalised at their tip by the adhesin PilC/PilY1. How this unusually large non-pilin protein is stably displayed at the pilus tip, and why is it required for pilus biogenesis, remains unknown. Here, using a multidisciplinary approach in Neisseria meningitidis, we show that the last 12 residues of PilC, representing ~1% of the protein, are both necessary and sufficient for pilus-tip localisation and filament biogenesis. X-ray crystallography reveals that this short "plug" motif binds the PilK subunit (within a complex of four minor pilins that caps the pilus) through {beta}-strand augmentation, a mode of interaction not previously characterised in T4F. Binding assays with synthetic peptides establish the specificity and affinity of this interaction, while the addition of a plug peptide extracellularly to cultures of a{Delta} pilC mutant restores pilus biogenesis. We show, using different methods, that the plug motif markedly stabilises PilK, providing an explanation for the requirement of PilC in pilus biogenesis. Consequently, expression of a PilK protein carrying a fused plug in N. meningitidis bypasses the requirement for PilC in pilus biogenesis. Together, these findings define the molecular basis of PilC/PilY1 function and localisation, reconcile all previous observations, and establish a broadly applicable model for T4aP biogenesis.

microbiology↗

bioRxiv2026

Cardiolipin constrains lipid unsaturation during anaerobic adaptation in Escherichia coli

Membrane lipids play a crucial role in cellular adaptation; though their specific functions in bacterial adaptation to oxygen limitation are not yet fully elucidated. Cardiolipin (CL), a signature phospholipid in energy-transducing membranes and a key organizer of mitochondrial respiration, has an unclear role in bacterial anaerobic physiology. Here, genetics, quantitative lipidomics and proteomics, enzymology, and fluorescence imaging were combined to explore how CL facilitates hypoxia adaptation in Escherichia coli. ClsA emerged as the predominant CL synthase under anaerobic conditions, and CL deficiency selectively impaired nitrate-dependent growth, while fermentation and fumarate respiration remained largely unaffected. CL depletion triggered extensive lipidome rewiring during the aerobic-to-anaerobic transition, including increased levels of phosphatidylglycerol, phosphatidic acid, and diacylglycerol, along with a broad enrichment of more unsaturated lipid species. In parallel, proteome remodeling linked CL loss to reduced abundance of proteins involved in anoxic respiration and nitrosative stress management, alongside the induction of membrane stress responses. Interestingly, CL deficiency did not markedly affect cell morphology, nor the spatial distribution or stability of respiratory complexes. This suggests a specialized role in optimizing membrane protein function rather than providing generic structural support. Wild-type lipidome profiling further showed that anaerobiosis induces a shift of lipid species toward higher unsaturation, with only modest class-level changes. Collectively, these results connect lipid remodeling to functional outcomes in vivo, offering mechanistic insights into how bacteria adapt their membranes to maintain energy conservation in fluctuating environments.

microbiology↗

bioRxiv2025

Tunable nucleofugality in carbamoyl-bearing covalent cholinesterase inhibitors

A handful of carbamate warheads is utilised in chemical biology to target serine hydrolases. The following case study on cholinesterases is the first comprehensive structure-reactivity exploration of the carbamoyl warhead, rather than one-target-oriented structure-activity study, with in-depth profiling of diverse halogen, chalcogen, and nitrogen-based leaving groups (nucleofuges) that can tune warhead reactivity. With computational tools we correlated the experimentally observed reactivities with steric and electronic factors of the investigated warheads. QM/MM simulations considering the enzymatic environment explained how substitution of carbon for nitrogen in the leaving groups of compounds 26 and 28 through resonance stabilisation, inductive bond polarization, and acidity amplification lowered the reaction barrier and increased the reaction rate >360 million times, making compound 28 a covalent inhibitor. Our findings underline the complexity of covalent inhibition and demonstrate that multiple complementary methods are required to interpret and predict covalent behaviour. Additionally, even though carbamates typically act as slow substrates, we were able to slow down decarbamoylation to a point where inhibition became de facto irreversible. The most interesting O-isoxazol-3-yl carbamate warhead was further profiled against the wider human proteome and showed low off-target reactivity, making it useful in further drug discovery. By establishing structure-reactivity principles for carbamoyl warhead, this study provides a generalisable framework for the development of selective covalent inhibitors and activity-based probes across diverse targets.

pharmacology and toxicology↗