Artur Gora

CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures

UNLABELLED The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graphic user interface for setting up the calculation and interactive exploration of identified tunnels/channels and their characteristics. AVAILABILITY AND IMPLEMENTATION CAVER Analyst is a multi-platform software written in JAVA. Binaries and documentation are freely available for non-commercial use at http://www.caver.cz.

Gates of Enzymes

This review highlights the importance of gates in enzymes. The gates control substrate access to the active site and product release, restrict solvent access to specific protein regions, and synchronize processes occurring in distinct parts of the enzyme. Survey of 129 gates in 71 enzymes enabled a rigorous definition of gates and establishment of a new scheme for their classification. Gates were assigned to six distinct classes – wings, swinging doors, apertures, drawbridges, double drawbridges and shells. Presented are summary statistics describing the propensity of specific amino acid residues in particular gate classes. The proposed classification scheme provides guidance for the analysis and engineering of gates in biomolecular systems.

Software tools for identification, visualization and analysis of protein tunnels and channels.

Protein structures contain highly complex systems of voids, making up specific features such as surface clefts or grooves, pockets, protrusions, cavities, pores or channels, and tunnels. Many of them are essential for the migration of solvents, ions and small molecules through proteins, and their binding to the functional sites. Analysis of these structural features is very important for understanding of structure-function relationships, for the design of potential inhibitors or proteins with improved functional properties. Here we critically review existing software tools specialized in rapid identification, visualization, analysis and design of protein tunnels and channels. The strengths and weaknesses of individual tools are reported together with examples of their applications for the analysis and engineering of various biological systems. This review can assist users with selecting a proper software tool for study of their biological problem as well as highlighting possible avenues for further development of existing tools. Development of novel descriptors representing not only geometry, but also electrostatics, hydrophobicity or dynamics, is needed for reliable identification of biologically relevant tunnels and channels.

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Background: Tunnel properties affect ligand passage in enzymes with buried active sites. Results: A tunnel mutation from leucine to tryptophan changes the mechanism of bromide ion release from haloalkane dehalogenase LinB. Conclusion: Interactions of the bromide ion with the tryptophan increase free energy barrier for its passage, causing the reaction mechanism change. Significance: The results provide guidelines for enzyme engineering. Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

Haloalkane dehalogenases catalyze the hydrolysis of carbon–halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide‐stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan–asparagine pair. The newly‐isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide‐stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre‐steady‐state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen‐bonding patterns within the active sites of the wild‐type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7‐fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K0.5 for selected halogenated substrates by a factor of 2–4, without impacting the rate‐determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

AQUA-DUCT: a ligands tracking tool

Motivation: The identification and tracking of molecules which enter active site cavity requires screening the positions of thousands of single molecules along several thousand molecular dynamic steps. To fill the existing gap between tools searching for tunnels and pathways and advanced tools employed for accelerated water flux investigations, we have developed AQUA‐DUCT. Results: AQUA‐DUCT is an easy‐to‐use tool that facilitates analysis of the behaviour of molecules that penetrate any selected region in a protein. It can be used for any type of molecules, e.g. water, oxygen, carbon dioxide, organic solvents, ions. Availability and Implementation: Linux, Windows, macOS, OpenBSD, http://www.aquaduct.pl. Contact: a.gora@tunnelinggroup.pl or info@aquaduct.pl Supplementary information: Supplementary data are available at Bioinformatics online.

Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

BALCONY: an R package for MSA and functional compartments of protein variability analysis

BackgroundHere, we present an R package for entropy/variability analysis that facilitates prompt and convenient data extraction, manipulation and visualization of protein features from multiple sequence alignments. BALCONY can work with residues dispersed across a protein sequence and map them on the corresponding alignment of homologous protein sequences. Additionally, it provides several entropy and variability scores that indicate the conservation of each residue.ResultsOur package allows the user to visualize evolutionary variability by locating the positions most likely to vary and to assess mutation candidates in protein engineering.ConclusionIn comparison to other R packages BALCONY allows conservation/variability analysis in context of protein structure with linkage of the appropriate metrics with physicochemical features of user choice.Availability: CRAN project page: https://cran.r-project.org/package=BALCONY and our website: http://www.tunnelinggroup.pl/software/ for major platforms: Linux/Unix, Windows and Mac OS X.