Biology subjects

Tokarz, P.

Publications and source records attributed to Tokarz, P..

3 recordsLinked to original sources

bioRxiv2024

Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of {beta}-(1[->]2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 [A] and 3.4 [A] resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

biochemistry↗

bioRxiv2024

Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE

Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 [A] and 3.5 [A], respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.

biochemistry↗

bioRxiv2022

SepN is essential for assembly and gating of septal junctions in Nostoc sp. PCC 7120

The multicellular life style of filamentous cyanobacteria like Nostoc sp. PCC7120 relies on a cell-cell communication system involving so called septal junctions. These are multiprotein complexes, which traverse the septal peptidoglycan through nanopores, connecting the neighboring cells and enabling molecule transfer along the filament. The intercellular communication is crucial when different cell types in the filament, vegetative cells and heterocysts, have to exchange metabolites and signaling molecules. Septal junctions of cyanobacteria can even control the molecule exchange by gating. The multiprotein complex consists of three modules: the septum spanning tube, the plug residing within the cytoplasmic membrane at both ends of the tube and a membrane associated cap module, covering the plug/tube modules on the cytoplasmic side of each neighboring cell. Until now, FraD was the only identified protein component of the septal junction protein complexes and localizes to the plug module. Here, we identified SepN as a new component via co-immunoprecipitation using FraD as bait and further demonstrated its essential role in septal junction assembly. Despite normal septal nanopore formation, a mutant in sepN exhibited a highly reduced rate of intercellular communication and was unable to gate the exchange of molecules. Cryo-electron tomography of cryo-focused ion beam thinned sepN-mutant filaments revealed septal junctions lacking the plug module and lateral cap openings. The combination of missing plug but present cap allowed to deduce the importance of the plug module in ensuring the correct cap architecture and, more importantly, in sealing the diffusion area in the closed septal junction state.

microbiology↗