FReDoWS: a method to automate molecular docking simulations with explicit receptor flexibility and snapshots selection

<p>Abstract</p> <p>Background</p> <p><it>In silico</it> molecular docking is an essential step in modern drug discovery when driven by a well defined macromolecular target. Hence, the process is called structure-based or rational drug design (RDD). In the docking step of RDD the macromolecule or receptor is usually considered a rigid body. However, we know from biology that macromolecules such as enzymes and membrane receptors are inherently flexible. Accounting for this flexibility in molecular docking experiments is not trivial. One possibility, which we call a fully-flexible receptor model, is to use a molecular dynamics simulation trajectory of the receptor to simulate its explicit flexibility. To benefit from this concept, which has been known since 2000, it is essential to develop and improve new tools that enable molecular docking simulations of fully-flexible receptor models.</p> <p>Results</p> <p>We have developed a Flexible-Receptor Docking Workflow System (FReDoWS) to automate molecular docking simulations using a fully-flexible receptor model. In addition, it includes a snapshot selection feature to facilitate acceleration the virtual screening of ligands for well defined disease targets. FReDoWS usefulness is demonstrated by investigating the docking of four different ligands to flexible models of <it>Mycobacterium tuberculosis’</it> wild type InhA enzyme and mutants I21V and I16T. We find that all four ligands bind effectively to this receptor as expected from the literature on similar, but wet experiments.</p> <p>Conclusions</p> <p>A work that would usually need the manual execution of many computer programs, and the manipulation of thousands of files, was efficiently and automatically performed by FReDoWS. Its friendly interface allows the user to change the docking and execution parameters. Besides, the snapshot selection feature allowed the acceleration of docking simulations. We expect FReDoWS to help us explore more of the role flexibility plays in receptor-ligand interactions. FReDoWS can be made available upon request to the authors.</p

The <it>Mycobacterium tuberculosis </it>Rv2540c DNA sequence encodes a bifunctional chorismate synthase

<p>Abstract</p> <p>Background</p> <p>The emergence of multi- and extensively-drug resistant <it>Mycobacterium tuberculosis </it>strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for <it>M. tuberculosis </it>and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the <it>M. tuberculosis </it>Rv2540c (<it>aroF</it>) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.</p> <p>Results</p> <p>In the present work, we describe DNA amplification of <it>aroF</it>-encoded CS from <it>M. tuberculosis </it>(<it>Mt</it>CS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant <it>Mt</it>CS. The bifunctionality of <it>Mt</it>CS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN_oxand <it>Mt</it>CS. FMN_oxand NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.</p> <p>Conclusion</p> <p>This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C₄-<it>proS </it>hydrogen is being transferred during the reduction of FMN_oxby NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMN_ox. The results on enzyme kinetics described here provide evidence for the mode of action of <it>Mt</it>CS and should thus pave the way for the rational design of antitubercular agents.</p

Timing it right: Precise ON/OFF switches for Rho1 and Cdc42 GTPases in cytokinesis

10.1083/jcb.201306152Journal of Cell Biology2022187-189JCLB