Biology subjects

Pedebos, C.

Publications and source records attributed to Pedebos, C..

6 recordsLinked to original sources

bioRxiv2022

Molecular architecture of the TasA biofilm scaffold in Bacillus subtilis

Many bacteria in nature exist in multicellular communities termed biofilms. Cells within biofilms are embedded in a primarily self-secreted extracellular polymeric matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model biofilm-forming bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. The structure of TasA fibres, however, and how fibres scaffold the biofilm at the molecular level, is not known. Here, we present electron cryomicroscopy structures of TasA fibres, which show that rather than forming amyloid fibrils, TasA monomers assemble into filaments through donor strand complementation, with each subunit donating a {beta}-strand to complete the fold of the next subunit along the filament. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA filaments congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. This study explains the previously observed biochemical properties of TasA and shows, for the first time, how a bacterial extracellular globular protein can assemble from monomers into {beta}-sheet-rich fibres, and how such fibres assemble into bundles in biofilms. We establish a hierarchical, atomic-level assembly mechanism of biofilm scaffolding that provides a structural framework for understanding bacterial biofilm formation.

microbiology↗

bioRxiv2021

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The recent global COVID-19 pandemic has prompted a rapid response in terms of vaccine and drug development targeting the viral pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we modelled a complete membrane-embedded SARS-CoV-2 spike (S) protein, the primary target of vaccine and therapeutics development, based on available structural data and known glycan content. We then used molecular dynamics (MD) simulations to study the system in the presence of benzene probes designed to enhance discovery of cryptic, potentially druggable pockets on the S protein surface. We uncovered a novel cryptic pocket with promising druggable properties located underneath the 617-628 loop, which was shown to be involved in the formation of S protein multimers on the viral surface. A marked multi-conformational behaviour of this loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments, supportive of opening and closing dynamics. Interestingly, the pocket is also the site of the D614G mutation, known to be important for SARS-CoV-2 fitness, and within close proximity to mutations in the novel SARS-CoV-2 strains B.1.1.7 and B.1.1.28, both of which are associated with increased transmissibility and severity of infection. The pocket was present in systems emulating both immature and mature glycosylation states, suggesting its druggability may not be dependent upon the stage of virus maturation. Overall, the predominantly hydrophobic nature of the cryptic pocket, its well conserved surface, and proximity to regions of functional relevance in viral assembly and fitness are all promising indicators of its potential for therapeutic targeting. Our method also successfully recapitulated hydrophobic pockets in the receptor binding domain and N-terminal domain associated with detergent or lipid binding in prior cryo-electron microscopy (cryo-EM) studies. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

biophysics↗

bioRxiv2021

Structural basis for silicon uptake by higher plants

Metalloids are elements with physical and chemical properties that are intermediate between metals and non-metals. Silicon (Si) is the most abundant metalloid in the Earths crust and occurs at high levels in many plants, especially those belonging to the Poaceae (grasses). Most of the worlds staple food crops such as rice, barley and maize accumulate silicon to high levels, resulting in resistance to abiotic and biotic stresses and consequently better plant growth and crop yields. The first step in silicon accumulation is the uptake of silicic acid (Si), the bioavailable from of silicon, by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins. Here we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). While the OsNIP2;1 channel is closed in the crystal by intracellular loop D, unbiased molecular dynamics (MD) simulations reveal a rapid channel opening on sub-microsecond time scales. MD simulations further show how Si interacts with an extracellular five-residue selectivity filter that provides the main barrier for transmembrane diffusion. Our data provide a foundation for understanding and potential manipulation of metalloid selectivity of an important and understudied aquaporin subfamily. SignificanceMany of the worlds most important food crops such as rice, barley and maize accumulate silicon to high levels, resulting in better plant growth and crop yields. The first step in silicon accumulation is the uptake of silicic acid (Si) by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins. Here, we present the X-ray crystal structure and molecular dynamics simulations of the archetypal NIP family member from Oryza sativa (OsNIP2;1) to visualise Si uptake. Our data provide a platform for improved understanding of Si uptake by plants that could be utilised, e.g., in silicon biofortification of important crops and potential alleviation of arsenic accumulation in the rice grain.

plant biology↗

bioRxiv2020

Assessment of Structural Units Deletions in the Archaeal Oligosaccharyltransferase AglB

Oligosaccharyltransferases (OSTs) are enzymes that catalyze the transfer of a glycan chain to an acceptor protein. Their structure is composed by a transmembrane domain and a periplasmic / C-terminal domain, which can be divided into structural units. The Archaeoglobus fulgidus OST, AfAglB, has unique structural units with unknown functions. Here, we evaluate the stability role proposed for AfAglB units by employing molecular modelling and molecular dynamics simulations, to examine the effect of single and double deletions in the enzyme structure. Our results show a strong effect on the dynamics of the C-terminal domain for the mutated systems with increased fluctuations near the deleted areas. Conformational profile and stability are deeply affected, mainly in the double unit deletion, modifying the enzyme behavior and binding interfaces. Coordination at the catalytic site was not disrupted, indicating that the mutated enzymes could retain activity at some level. Hotspots of variation were identified and rationalized with previous data. Our data shows that structural units may provide stabilization interactions, contributing for integrity of the wild-type enzyme at high temperatures. By correlating our findings to structural units mutagenesis experimental data available, it was observed that structural units deletion can interfere with OSTs stability and dynamics but it is not directly related to catalysis. Instead, they may influence the OST structural integrity, and, potentially, thermostability. This work offers a basis for future experiments involving OSTs structural and functional characterization, as well as for protein engineering.

biophysics↗

bioRxiv2020

The Hitchhiker's Guide to the Periplasm: Unexpected Molecular Interactions of Antibiotics Revealed by Considering Crowding Effects in E. coli

The periplasm of Gram-negative bacteria is a highly crowded environment comprised of many different molecular species. Antibacterial agents that causes lysis of Gram-negative bacteria by their action against the inner membrane must cross the periplasm to arrive at their target membrane. Very little is currently known about their route through the periplasm, and the interactions they experience. To this end, here atomistic molecular dynamics simulations are used to study the path taken by the antibiotic polymyxin B1 through a number of models of the periplasm which are crowded with proteins and osmolytes to different extents. The simulations reveal that PMB1 forms transient and long-lived interactions with proteins and osmolytes that are free in solution as well as lipoproteins anchored to the outer membrane and bound to the cell wall. We show that PMB1 may be able to hitchhike within the periplasm by binding to lipoprotein carriers. Overall our results show that PMB1 is rarely uncomplexed within the periplasm; an important consideration for interpretations of its therapeutic mechanism of action. It is likely that this observation can be extended to other antibiotics that rely on diffusion to cross the periplasm.

biochemistry↗

bioRxiv2019

Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3

Genetic variation in the enzymes that catalyse post-translational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signalling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme that acts as a ubiquitin donor for E3 ligases that catalyse ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologues to identify a position in the C-terminal -helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse ES cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal in mice. When mutant S138A ESCs were differentiated into extra-embryonic primitive endoderm (PrE), levels of the PDGFR and FGFR1 receptor tyrosine kinases (RTKs) were reduced and PreE differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the RTKs. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.

developmental biology↗