Characterising ATP binding activity of PilB ATPase of Shewanella oneidensis MR-1 using a molecular modeling and simulations approach.

This study characterises the interaction between the type IV pilus assembly PilB ATPase of a versatile electroactive microbe, Shewanella oneidensis MR-1, and ATP using in silico tools. PilB ATPase, which is associated with different cellular activities, is a protein subunit of type IV pili. A composite model of the protein was generated using the I-TASSER Web server and its stereochemical quality was evaluated using PROCHECK. Loop modeling was performed using the InteractiveRosetta tool to refine the structure of the model and the COACH server was used to identify the functional binding site. The nature of binding, with the native ligand ATP, was determined using Autodock Vina and Discovery Studio Visualizer. Molecular dynamics simulations were carried out, with the bound and unbound states of the protein, for a period of 100 ns using GROMACS. Favorable root mean square deviation (0.75±0.10 nm) and radius of gyration (2.78±0.05 nm) values pointed to the stability of the modeled protein structure. Root mean square fluctuation and solvent accessible surface area analyses indicated a conformational change upon the ligand binding which occurred without a corresponding reorganization of secondary structures as evidenced by definition of secondary protein analysis. Molecular mechanics/Poisson-Boltzmann surface area analysis revealed the presence of a loop critical to the formation of stable interactions with ATP.

Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes.

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based in vitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.

Metamorphism in TDP-43 prion-like domain determines chaperone recognition.

The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and co-chaperones primarily recognize the structured elements in TDP-43´s prion-like domain. Significantly, while HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in disease.

Beneficial Biofilms: a Minireview of Strategies To Enhance Biofilm Formation for Biotechnological Applications.

The capacity of bacteria to form biofilms is an important trait for their survival and persistence. Biofilms occur naturally in soil and aquatic environments, are associated with animals ranging from insects to humans, and are also found in built environments. They are typically encountered as a challenge in health care, food industry, and water supply ecosystems. In contrast, they are known to play a key role in the industrial production of commercially valuable products, environmental remediation processes, and microbe-catalyzed electrochemical systems for energy and resource recovery from wastewater. While there are many recent articles on biofilm control and removal, review articles on promoting biofilm growth for biotechnological applications are unavailable. Biofilm formation is a tightly regulated response to perturbations in the external environment. The multistage process, mediated by an assortment of proteins and signaling systems, involves the attachment of bacterial cells to a surface followed by their aggregation in a matrix of extracellular polymeric substances. Biofilms can be promoted by altering the external environment in a controlled manner, supplying molecules that trigger the aggregation of cells and engineering genes associated with biofilm development. This minireview synthesizes findings from studies that have described such strategies and highlights areas needing research attention.

Identifying potential inhibitors of biofilm-antagonistic proteins to promote biofilm formation: a virtual screening and molecular dynamics simulations approach.

Microbial biofilms play a critical role in environmental biotechnology and associated applications. Biofilm production can be enhanced by inhibiting the function of proteins that negatively regulate their formation. With this objective, an in silico approach was adopted to identify competitive inhibitors of eight biofilm-antagonistic proteins, namely AbrB and SinR (from Bacillus subtilis) and AmrZ, PDE (EAL), PslG, RetS, ShrA and TpbA (from Pseudomonas aeruginosa). Fifteen inhibitors that structurally resembled the natural ligand of each protein were shortlisted using ligand-based and structure-based virtual screening. The top four inhibitors obtained from molecular docking using Autodock Vina were further docked using SwissDock and DOCK 6.9 to obtain a consensus hit for each protein based on different scoring functions. Further analysis of the protein-ligand complexes revealed that these top inhibitors formed significant non-covalent interactions with their respective protein binding sites. The eight protein-ligand complexes were then subjected to molecular dynamics simulations for 30 ns using GROMACS. RMSD and radius of gyration values of 0.1-0.4 nm and 1.0-3.5 nm, respectively, along with hydrogen bond formation throughout the trajectory indicated that all the complexes remained stable, compact and intact during the simulation period. Binding energy values between -20 and -77 kJ/mol obtained from MM-PBSA calculations further confirmed the high affinities of the eight inhibitors for their respective receptors. The outcome of this study holds great promise to enhance biofilms that are central to biotechnological processes associated with microbial electrochemical technologies, wastewater treatment, bioremediation and the industrial production of value-added products.