Biology subjects

Ung, V.

Publications and source records attributed to Ung, V..

4 recordsLinked to original sources

bioRxiv2025

Protease mimicry: dissecting the ester bond crosslinking mechanics in bacterial adhesin proteins

The ester bond crosslink discovered within bacterial adhesin proteins offers a captivating insight into the convergent evolution of enzyme-like machinery. Crystal structures reveal a putative catalytic triad comprising an acid-base-nucleophile combination and an oxyanion-like site that suggest a serine protease-like mechanism drives the crosslinking process. We now provide confirmation of the mechanism, revealing functional catalytic dyads or triads, and the recapitulation of protease machinery from a Pseudomonas bacterium and a human cytomegalovirus related only by convergent evolution. Molecular dynamics simulations show how a conservative threonine-to-serine mutation of the nucleophile induces hydrolysis and eliminates the ester bond crosslink. Collectively, our structural, functional, and computational efforts detail the molecular intricacies of intramolecular ester bond formation and underscore the convergent evolutionary adaptations of bacteria in exploiting enzyme-like machinery to protect essential adhesin proteins from the mechanical, biological, and chemical hostilities of the bacterias replicative niche.

biochemistry↗

bioRxiv2024

Disordered regions of human eIF4B orchestrate a dynamic self-association landscape

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

biophysics↗

bioRxiv2022

The structural, dynamical and biochemical characterizations of Verticillium dahliae pectate lyase, VdPelB, highlight its specificities

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by {beta}-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae, a plant pathogen. We first solved the crystal structure of VdPelB at 1.2[A] resolution showing that it is a right-handed parallel {beta}-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. VdPelB was most active on non-methylesterified pectins, at pH 8 in presence of Ca2+ ions. VdPelB-G125R mutant was most active at pH 9 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

biochemistry↗

bioRxiv2022

Differences in the structure of plant polygalacturonases specify enzymes dynamics and processivities to fine-tune pectins and root development

The fine-tuning of pectins by polygalacturonases (PGs) plays a key role in modulating plant cell wall chemistry and mechanics, impacting plant development. In plants, the high number of PGs encoded in the genome questions the regulation of pectin depolymerization and the roles of distinct isozymes in the control of development. Here we report the first crystal structures of two PGs from Arabidopsis, PGLR and ADPG2 whose expression overlap in roots. Albeit having overall conserved folds and active sites, PGLR and ADPG2 differed in the structure of their binding grooves and in the amino-acids of the subsites. We determined the structural features that explain the absence of inhibition of the plant PGs by endogenous PG-Inhibiting Proteins (PGIPs). By combining molecular dynamic simulations, analysis of enzymes kinetics and hydrolysis products, we showed that subtle differences in PGLR and ADPG2 structures translated into distinct enzyme-substrate dynamics and enzymes processivities. Using the plant root as a developmental model, exogenous application of purified enzymes showed that these distinct PGLR/ADPG2 processivities ultimately translated into different impacts on development. The highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. Our study suggests that, in plants, gene redundancy is unlikely to reflect redundant biochemical specificities. Isozymes of distinct specificities and processivities are likely to be of major importance for the fine spatial and temporal regulation of pectin structure. Significance StatementPlant polygalacturonases (PG) are enzymes that play a key role in the regulation of cell wall pectin chemistry by controlling the degree of polymerization of the HG chains. The high number of genes encoding PG in Arabidopsis questions the rationale for such abundance. We solved the crystal structure of two PG (PGLR and ADPG2) whose expression overlap in roots and showed, using combined computational and experimental approaches, that they differ in their enzyme-substrate dynamics, leading to distinct processivities. The highly processive ADPG2 can generate digestion products of shorter degree of polymerization, and upon exogenous application on developing roots, induced drastic developmental defects. Our study suggests that gene redundancy is unlikely to reflect redundant biochemical specificities of isozymes.

biochemistry↗