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AlphaFold - a game changer for accurate protein structure prediction was tested on metamorphic proteins, which display two or more distinct folds while having the same sequence. We highlighted the limitations of the method and suggested careful investigation of obtained models.

Graphical abstract, Highlights • Lineage of a sEH determines its overall flexibility and tunnels architecture. • sEHs flexibility and tunnel network regulate substrate positioning. • Extensive tunnel network can be substantial for hydrolytic specificity. • sEHs predominantly utilize one tunnel or both of them. • Rearrangements in protein tunnel network can be caused by fragment insertions., Enzymes with buried active sites maintain their catalytic function via a single tunnel or tunnel network. In this study we analyzed the functionality of soluble epoxide hydrolases (sEHs) tunnel network, by comparing the overall enzyme structure with the tunnel’s shape and size. sEHs were divided into three groups based on their structure and the tunnel usage. The obtained results were compared with known substrate preferences of the studied enzymes, as well as reported in our other work evolutionary analyses data. The tunnel network architecture corresponded well with the evolutionary lineage of the source organism and large differences between enzymes were observed from long fragments insertions. This strategy can be used during protein re-engineering process for large changes introduction, whereas tunnel modification can be applied for fine-tuning of enzyme.

The evolutionary variability of a protein’s residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/β-hydrolase fold proteins - one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels’ evolution analysis based on entropy values calculated for tunnels’ residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a ‘perforation’ mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme’s performance and can be applied widely for enzymes with buried active sites.Author SummarySo far very little is known about proteins tunnels evolution. The goal of this study is to evaluate the evolution of tunnels in the family of soluble epoxide hydrolases - representatives of numerous α/β-hydrolase fold enzymes. As a result two types of tunnels evolution analysis were proposed (a general and a detailed approach), as well as a ‘perforation’ mechanism which can mimic native evolution in proteins and can be used as an additional strategy for enzymes redesign.

In the present study we tested, using the microscale thermophoresis technique, a small library of thionocarbamates, thiolocarbamates, sulfide and disulfide as potential lead compounds for SARS-CoV-2 Mpro drug design. The successfully identified binder is a representative of the thionocarbamates group with a high potential for future modifications aiming for higher affinity and solubility. The experimental analysis was extended by computational studies that show insufficient accuracy of the simplest and widely applied approaches and underline the necessity of applying more advanced methods to properly evaluate the affinity of potential SARS-CoV-2 Mpro binders.

Human soluble epoxide hydrolase (hsEH) is involved in the hydrolysis of epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory properties. Given that EET conversion generates nonbioactive molecules, inhibition of this enzyme would be beneficial. Past decades of work on hsEH inhibitors resulted in numerous potential compounds, of which a hundred hsEH-ligand complexes were crystallized and deposited in the Protein Data Bank (PDB). We analyzed all deposited hsEH-ligand complexes to gain insight into the binding of inhibitors and to provide feedback on the future drug design processes. We also reviewed computationally driven strategies that were used to propose novel hsEH inhibitors.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (Mpro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 Mpro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV Mpro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 Mpro inhibitors, as we report it, was not previously conducted.

(1) Background: Molybdenum cofactor deficiency type B (MOCODB, #252160) is a rare autosomal recessive metabolic disorder characterized by intractable seizures of neonatal-onset, muscular spasticity, accompanying with hypouricemia, elevated urinary sulfite levels and craniofacial dysmorphism. Thirty-five patients were reported to date. (2) Methods: Our paper aimed to delineate the disease genotype by presenting another patient, in whom a novel, in-frame variant within the MOCS2 gene was identified. (3) Results: Exome sequencing led to the identification of a novel variant in the MOCS2 gene-c.472_477del of unknown significance (VUS). (4) Conclusions: To prove the clinical significance of the mentioned variant, analysis of the possible mutation consequences on molecular level with the use of the available crystal structure of the human molybdopterin synthase complex was of great importance. Moreover, a potential pathomechanism resulting from a molecular defect was presented, giving original insight into the current knowledge on this rare disease, including treatment options.

Molybdenum cofactor deficiency type B (MOCODB, #252160) is a rare autosomal recessive metabolic disorder characterized by intractable seizures of neonatal-onset, muscular spasticity, accompanying with hypouricemia, elevated urinary sulfite levels and craniofacial dysmorphism. Thirty-five patients were reported to date. Our paper aimed to delineate the disease genotype by presenting another patient, in whom novel, inframe variant within the MOCS2 gene was identified. Its clinical significance was supported by the medical history and analysis of the possible mutation consequences on a molecular level with the use of the available crystal structure of the human molybdopterin synthase complex. Moreover, potential pathomechanism resulting from molecular defect was presented, giving original insight into current knowledge on this rare disease, including treatment options.

Human soluble epoxide hydrolase (hsEH) has been known to be involved in the hydrolysis of epoxyeicosatrienoic acids (EETs), which are anti-inflammatory and cardioprotective signalling molecules. Since the conversion of EETs into the corresponding diols generates non-bioactive molecules, the enzyme’s inhibition would be beneficial. hsEH quickly became the molecular target, however, the proposed inhibitors directed toward this enzyme have not undergone clinical trials, mostly due to their low solubility in water. Our goal is to indicate new regions, distinct from those already investigated, that could be considered during the development of novel and perhaps more soluble inhibitors of hsEH. To fulfil this goal, we used a combination of classical and mixed-solvent molecular dynamics (cMD and MixMD) simulations and a ligand tracking approach. Such a strategy, considers conformational changes in the interior of the enzyme, extends conformational space of binding cavities, and provides insight into different sets of potential binding modes. By analysis of the local distribution of different molecular probes (water and organic cosolvent molecules), we were able to identify potential binding sites (hot-spots), which can indicate the location of the most important functional groups for the binding of designed inhibitors. The uniqueness of the binding spots was examined by superpositioning the indicated hot-spots with a map of chemical interactions between the known inhibitors and hsEH. We analysed all available crystal structures of hsEHs with inhibitors deposited in the PDB database to gain insight into the binding modes of the known inhibitors and to find similarities and dissimilarities between them. By comparing the results obtained based on the map of chemical interactions, with the regions identified by using cMD and MixMD, we were able to identify regions that have not been targeted by ligands until now and which are promising possible binding sites for polar groups that would be beneficial for inhibitors solubility.

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site&rsquo, s conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus&rsquo, mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

AQUA-DUCT software reverses the standard approach of the molecular dynamics simulations analysis of macromolecules, focusing on solvent, cosolvent and small ligands analysis considered as specific molecular probes instead of analysis of macromolecules atoms movement. In this article we present six basic tutorials instructing the users in the best practices for preparing, carrying out, and analysing AQUA-DUCT results in various applications. Users are expected to already have significant experience with running standard molecular dynamics simulations in any dedicated software (e.g., Amber, GROMACS, NAMD), and usage of PyMOL visualising software. The tutorials range from a basic analysis of multiple solvents trajectory used for identification of the entries/exits to the protein core, to a complex one, like identification of the key hot-spots in cosolvent MD simulations that can be used as an insight for macromolecules description, analysis, re-engineering, and for drug design.

Motivation Tunnels, pores, channels, pockets and cavities contribute to proteins architecture and performance. However, analysis and characteristics of transportation pathways and internal binding cavities are performed separately. We aimed to provide universal tool for analysis of proteins integral interior with access to detailed information on the ligands transportation phenomena and binding preferences. Results AQUA-DUCT version 1.0 is a comprehensive method for macromolecules analysis from the intramolecular voids perspective using small ligands as molecular probes. This version gives insight into several properties of macromolecules and facilitates protein engineering and drug design by the combination of the tracking and local mapping approach to small ligands. Availability and implementation http://www.aquaduct.pl. Supplementary information Supplementary data are available at Bioinformatics online.

Transport of ligands between buried active sites and bulk solvent is a key step in the catalytic cycle of many enzymes. The absence of evolutionary optimized transport tunnels is an important barrier limiting the efficiency of biocatalysts prepared by computational design. Creating a structurally defined and functional “hole” into the protein represents an engineering challenge. Here we describe the computational design and directed evolution of a de novo transport tunnel in haloalkane dehalogenase. Mutants with a blocked native tunnel and newly opened auxiliary tunnel in a distinct part of the structure showed dramatically modified properties. The mutants with blocked tunnels acquired specificity never observed with native family members: up to 32 times increased substrate inhibition and 17 times reduced catalytic rates. Opening of the auxiliary tunnel resulted in specificity and substrate inhibition similar to those of the native enzyme and the most proficient haloalkane dehalogenase reported to date (k...

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone. Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium. The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram'-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample. In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

Loops are the most variable and unorganized elements of the secondary structure of proteins. Their ability to shift their shape can play a role in the binding of small ligands, enzymatic catalysis, or protein&ndash, protein interactions. Due to the loop flexibility, the positions of their residues in solved structures show the largest B-factors, or in a worst-case scenario can be unknown. Based on the loops&rsquo, movements&rsquo, timeline, they can be divided into slow (static) and fast (flexible). Although most of the loops that are missing in experimental structures belong to the flexible loops group, the computational tools for loop reconstruction use a set of static loop conformations to predict the missing part of the structure and evaluate the model. We believe that these two loop types can adopt different conformations and that using scoring functions appropriate for static loops is not sufficient for flexible loops. We showed that common model evaluation methods, are insufficient in the case of flexible solvent-exposed loops. Instead, we recommend using the potential energy to evaluate such loop models. We provide a novel model selection method based on a set of geometrical parameters to distinguish between flexible and static loops without the use of molecular dynamics simulations. We have also pointed out the importance of water network and interactions with the solvent for the flexible loop modeling.

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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.

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 K(0.5) 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.

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.

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. Supplementary information Supplementary data are available at Bioinformatics online.

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.