Exploiting enzyme evolution for computational protein design.

Recent years have seen an explosion of interest in understanding the physicochemical parameters that shape enzyme evolution, as well as substantial advances in computational enzyme design. This review discusses three areas where evolutionary information can be used as part of the design process: (i) using ancestral sequence reconstruction (ASR) to generate new starting points for enzyme design efforts; (ii) learning from how nature uses conformational dynamics in enzyme evolution to mimic this process in silico; and (iii) modular design of enzymes from smaller fragments, again mimicking the process by which nature appears to create new protein folds. Using showcase examples, we highlight the importance of incorporating evolutionary information to continue to push forward the boundaries of enzyme design studies.

PredictONCO: a web tool supporting decision-making in precision oncology by extending the bioinformatics predictions with advanced computing and machine learning.

PredictONCO 1.0 is a unique web server that analyzes effects of mutations on proteins frequently altered in various cancer types. The server can assess the impact of mutations on the protein sequential and structural properties and apply a virtual screening to identify potential inhibitors that could be used as a highly individualized therapeutic approach, possibly based on the drug repurposing. PredictONCO integrates predictive algorithms and state-of-the-art computational tools combined with information from established databases. The user interface was carefully designed for the target specialists in precision oncology, molecular pathology, clinical genetics and clinical sciences. The tool summarizes the effect of the mutation on protein stability and function and currently covers 44 common oncological targets. The binding affinities of Food and Drug Administration/ European Medicines Agency -approved drugs with the wild-type and mutant proteins are calculated to facilitate treatment decisions. The reliability of predictions was confirmed against 108 clinically validated mutations. The server provides a fast and compact output, ideal for the often time-sensitive decision-making process in oncology. Three use cases of missense mutations, (i) K22A in cyclin-dependent kinase 4 identified in melanoma, (ii) E1197K mutation in anaplastic lymphoma kinase 4 identified in lung carcinoma and (iii) V765A mutation in epidermal growth factor receptor in a patient with congenital mismatch repair deficiency highlight how the tool can increase levels of confidence regarding the pathogenicity of the variants and identify the most effective inhibitors. The server is available at https://loschmidt.chemi.muni.cz/predictonco.

LoopGrafter: a web tool for transplanting dynamical loops for protein engineering.

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

Virtual screening of potential anticancer drugs based on microbial products.

The development of microbial products for cancer treatment has been in the spotlight in recent years. In order to accelerate the lengthy and expensive drug development process, in silico screening tools are systematically employed, especially during the initial discovery phase. Moreover, considering the steadily increasing number of molecules approved by authorities for commercial use, there is a demand for faster methods to repurpose such drugs. Here we present a review on virtual screening web tools, such as publicly available databases of molecular targets and libraries of ligands, with the aim to facilitate the discovery of potential anticancer drugs based on microbial products. We provide an entry-level step-by-step description of the workflow for virtual screening of microbial metabolites with known protein targets, as well as two practical examples using freely available web tools. The first case presents a virtual screening study of drugs developed from microbial products using Caver Web, a web tool that performs docking along a tunnel. The second case comprises a comparative analysis between a wild type isocitrate dehydrogenase 1 and a mutant that results in cancer, using the recently developed web tool PredictSNPOnco. In summary, this review provides the basic and essential background information necessary for virtual screening experiments, which may accelerate the discovery of novel anticancer drugs.

Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport.

Caver Web 1.0 is a web server for comprehensive analysis of protein tunnels and channels, and study of the ligands' transport through these transport pathways. Caver Web is the first interactive tool allowing both the analyses within a single graphical user interface. The server is built on top of the abundantly used tunnel detection tool Caver 3.02 and CaverDock 1.0 enabling the study of the ligand transport. The program is easy-to-use as the only required inputs are a protein structure for a tunnel identification and a list of ligands for the transport analysis. The automated guidance procedures assist the users to set up the calculation in a way to obtain biologically relevant results. The identified tunnels, their properties, energy profiles and trajectories for ligands' passages can be calculated and visualized. The tool is very fast (2-20 min per job) and is applicable even for virtual screening purposes. Its simple setup and comprehensive graphical user interface make the tool accessible for a broad scientific community. The server is freely available at https://loschmidt.chemi.muni.cz/caverweb.

Trimethylphosphate and Dimethylphosphate Hydrolysis by Binuclear CdII , MnII , and ZnII -FeII Promiscuous Organophosphate-Degrading Enzyme: Reaction Mechanisms.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 exhibits promiscuity, not only in the reactions it catalyzes, but also in the metals it uses to catalyze those reactions. Here, three different binuclear metal centers were studied: di-CdII , di-MnII and ZnII -FeII . This enzyme uses these metal centers for hydrolyzing trimethyl- and dimethyl-phosphates. Both mechanisms were studied at DFT level of theory using a cluster model approach. The ground spin state was determined for each enzyme. The outcomes confirmed that the hydrolysis of phosphotriester is faster than that of phosphodiester and in some case the phosphodiesterase reaction does not occur. The computed activation energies are in agreement with previous experimental results for phosphotriesterase enzymes.

Triesterase and promiscuous diesterase activities of a di-Co(II)-containing organophosphate degrading enzyme reaction mechanisms.

The reaction mechanism for the hydrolysis of trimethyl phosphate and of the obtained phosphodiester by the di-Co(II) derivative of organophosphate degrading enzyme from Agrobacterium radiobacter P230(OpdA), have been investigated at density functional level of theory in the framework of the cluster model approach. Both mechanisms proceed by a multistep sequence and each catalytic cycle begins with the nucleophilic attack by a metal-bound hydroxide on the phosphorus atom of the substrate, leading to the cleavage of the phosphate-ester bond. Four exchange-correlation functionals were used to derive the potential energy profiles in protein environments. Although the enzyme is confirmed to work better as triesterase, as revealed by the barrier heights in the rate-limiting steps of the catalytic processes, its promiscuous ability to hydrolyze also the product of the reaction has been confirmed. The important role played by water molecules and some residues in the outer coordination sphere has been elucidated, while the binuclear Co(II) center accomplishes both structural and catalytic functions. To correctly describe the electronic configuration of the d shell of the metal ions, high- and low-spin arrangement jointly with the occurrence of antiferromagnetic coupling, have been herein considered.

New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models.

Tyrosine ammonia lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid in purple phototropic bacteria and Actinomycetales. The enzyme is used in bioengineering and has the potential to be used industrially. It belongs to a family of enzymes that uses a 4-methylidene-imidazole-5-one (MIO) cofactor to catalyze the deamination amino acids. In the present work, we used a QM/MM and a QM cluster models of TAL to explore two putative reaction paths for its catalytic mechanism. Part of the N-MIO mechanism was previously studied by computational methods. We improved on previous studies by using a larger, more complete model of the enzyme, and by describing the complete reaction path. The activation energy for this mechanism, in agreement with the previous study, is 28.5 kcal/mol. We also found another reaction path that has overall better kinetics and reaches the products in a single reaction step. The barrier for this Single-Step mechanism is 16.6 kcal/mol, which agrees very well with the experimental kcat of 16.0 kcal/mol. The geometrical parameters obtained for the cluster and QM/MM models are very similar, despite differences in the relative energies. This means that both approaches are capable of describing the correct catalytic path of TAL.

A computational workflow for analysis of missense mutations in precision oncology.

Every year, more than 19 million cancer cases are diagnosed, and this number continues to increase annually. Since standard treatment options have varying success rates for different types of cancer, understanding the biology of an individual's tumour becomes crucial, especially for cases that are difficult to treat. Personalised high-throughput profiling, using next-generation sequencing, allows for a comprehensive examination of biopsy specimens. Furthermore, the widespread use of this technology has generated a wealth of information on cancer-specific gene alterations. However, there exists a significant gap between identified alterations and their proven impact on protein function. Here, we present a bioinformatics pipeline that enables fast analysis of a missense mutation's effect on stability and function in known oncogenic proteins. This pipeline is coupled with a predictor that summarises the outputs of different tools used throughout the pipeline, providing a single probability score, achieving a balanced accuracy above 86%. The pipeline incorporates a virtual screening method to suggest potential FDA/EMA-approved drugs to be considered for treatment. We showcase three case studies to demonstrate the timely utility of this pipeline. To facilitate access and analysis of cancer-related mutations, we have packaged the pipeline as a web server, which is freely available at https://loschmidt.chemi.muni.cz/predictonco/ .Scientific contributionThis work presents a novel bioinformatics pipeline that integrates multiple computational tools to predict the effects of missense mutations on proteins of oncological interest. The pipeline uniquely combines fast protein modelling, stability prediction, and evolutionary analysis with virtual drug screening, while offering actionable insights for precision oncology. This comprehensive approach surpasses existing tools by automating the interpretation of mutations and suggesting potential treatments, thereby striving to bridge the gap between sequencing data and clinical application.

Comparative analysis of the performance of commonly available density functionals in the determination of geometrical parameters for copper complexes.

In this study, a set of 50 transition-metal complexes of Cu(I) and Cu(II), were used in the evaluation of 18 density functionals in geometry determination. In addition, 14 different basis sets were considered, including four commonly used Pople's all-electron basis sets; four basis sets including popular types of effective-core potentials: Los Alamos, Steven-Basch-Krauss, and Stuttgart-Dresden; and six triple-ζ basis sets. The results illustrate the performance of different methodological alternatives for the treatment of geometrical properties in relevant copper complexes, pointing out Double-Hybrid (DH) and Long-range Correction (LC) Generalized Gradient Approximation (GGA) methods as better descriptors of the geometry of the evaluated systems. These however, are associated with a computational cost several times higher than some of the other methods employed, such as the M06 functional, which has also demonstrated a comparable performance. Regarding the basis sets, 6-31+G(d) and 6-31+G(d,p) were the best performing approaches. In addition, the results show that the use of effective-core potentials has a limited impact, in terms of the accuracy in the determination of metal-ligand bond-lengths and angles in our dataset of copper complexes. Hence, these could become a good alternative for the geometrical description of these systems, particularly CEP-121G and SDD basis sets, if one is considering larger copper complexes where the computational cost could be an issue.

Loop dynamics and the evolution of enzyme activity.

In the early 2000s, Tawfik presented his 'New View' on enzyme evolution, highlighting the role of conformational plasticity in expanding the functional diversity of limited repertoires of sequences. This view is gaining increasing traction with increasing evidence of the importance of conformational dynamics in both natural and laboratory evolution of enzymes. The past years have seen several elegant examples of harnessing conformational (particularly loop) dynamics to successfully manipulate protein function. This Review revisits flexible loops as critical participants in regulating enzyme activity. We showcase several systems of particular interest: triosephosphate isomerase barrel proteins, protein tyrosine phosphatases and ß-lactamases, while briefly discussing other systems in which loop dynamics are important for selectivity and turnover. We then discuss the implications for engineering, presenting examples of successful loop manipulation in either improving catalytic efficiency, or changing selectivity completely. Overall, it is becoming clearer that mimicking nature by manipulating the conformational dynamics of key protein loops is a powerful method of tailoring enzyme activity, without needing to target active-site residues.

Q-RepEx: A Python pipeline to increase the sampling of empirical valence bond simulations.

The exploration of chemical systems occurs on complex energy landscapes. Comprehensively sampling rugged energy landscapes with many local minima is a common problem for molecular dynamics simulations. These multiple local minima trap the dynamic system, preventing efficient sampling. This is a particular challenge for large biochemical systems with many degrees of freedom. Replica exchange molecular dynamics (REMD) is an approach that accelerates the exploration of the conformational space of a system, and thus can be used to enhance the sampling of complex biomolecular processes. In parallel, the empirical valence bond (EVB) approach is a powerful approach for modeling chemical reactivity in biomolecular systems. Here, we present an open-source Python-based tool that interfaces with the Q simulation package, and increases the sampling efficiency of the EVB free energy perturbation/umbrella sampling approach by means of REMD. This approach, Q-RepEx, both decreases the computational cost of the associated REMD-EVB simulations, and opens the door to more efficient studies of biochemical reactivity in systems with significant conformational fluctuations along the chemical reaction coordinate.

Screening of world approved drugs against highly dynamical spike glycoprotein of SARS-CoV-2 using CaverDock and machine learning.

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform virtual screening against spike glycoprotein using a cryogenic electron microscopy structure (PDB-ID: 6VXX) and the representative structures of five most populated clusters from a previously published molecular dynamics simulation. The dataset of ligands was obtained from the ZINC database and consists of drugs approved for clinical use worldwide. Trajectories for the passage of individual drugs through the tunnel of the spike glycoprotein homotrimer, their binding energies within the tunnel, and the duration of their contacts with the trimer's three subunits were computed for the full dataset. Multivariate statistical methods were then used to establish structure-activity relationships and select top candidate for movement inhibition. This new protocol for the rapid screening of globally approved drugs (4359 ligands) in a multi-state protein structure (6 states) showed high robustness in the rate of finished calculations. The protocol is universal and can be applied to any target protein with an experimental tertiary structure containing protein tunnels or channels. The protocol will be implemented in the next version of CaverWeb (https://loschmidt.chemi.muni.cz/caverweb/) to make it accessible to the wider scientific community.

LoopGrafter: Visual Support for the Grafting Workflow of Protein Loops.

In the process of understanding and redesigning the function of proteins in modern biochemistry, protein engineers are increasingly focusing on the exploration of regions in proteins called loops. Analyzing various characteristics of these regions helps the experts to design the transfer of the desired function from one protein to another. This process is denoted as loop grafting. As this process requires extensive manual treatment and currently there is no proper visual support for it, we designed LoopGrafter: a web-based tool that provides experts with visual support through all the loop grafting pipeline steps. The tool is logically divided into several phases, starting with the definition of two input proteins and ending with a set of grafted proteins. Each phase is supported by a specific set of abstracted 2D visual representations of loaded proteins and their loops that are interactively linked with the 3D view onto proteins. By sequentially passing through the individual phases, the user is shaping the list of loops that are potential candidates for loop grafting. In the end, the actual in-silico insertion of the loop candidates from one protein to the other is performed and the results are visually presented to the user. In this way, the fully computational rational design of proteins and their loops results in newly designed protein structures that can be further assembled and tested through in-vitro experiments. LoopGrafter was designed in tight collaboration with protein engineers, and its final appearance reflects many testing iterations. We showcase the contribution of LoopGrafter on a real case scenario and provide the readers with the experts' feedback, confirming the usefulness of our tool.

Computational design of enzymes for biotechnological applications.

Enzymes are the natural catalysts that execute biochemical reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial production of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technological advances have greatly facilitated this process by providing the experimental approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous laboratory work and economic resources. Theoretical methods provide predictions and represent an attractive complement to such experiments by waiving their inherent costs. Computational techniques can be used to engineer enzymatic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, solubility, and flexibility. Theoretical approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.

Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

Transcription and Translation Inhibitors in Cancer Treatment.

Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors. There are numerous druggable proteins involved in transcription and translation which make lucrative targets for cancer drug development. In addition to proteins, recent years have shown that the "undruggable" transcription factors and RNA molecules can also be targeted to hamper the transcription or translation in cancer. In this review, we summarize the properties and function of the transcription and translation inhibitors that have been tested and developed, focusing on the advances of the last 5 years. To complement this, we also discuss some of the recent advances in targeting oncogenes tightly controlling transcription including transcription factors and KRAS. In addition to natural and synthetic compounds, we review DNA and RNA based approaches to develop cancer drugs. Finally, we conclude with the outlook to the future of the development of transcription and translation inhibitors.

The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step.

Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.

Engineering enzyme access tunnels.

Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes.

Fast Screening of Inhibitor Binding/Unbinding Using Novel Software Tool CaverDock.

Protein tunnels and channels are attractive targets for drug design. Drug molecules that block the access of substrates or release of products can be efficient modulators of biological activity. Here, we demonstrate the applicability of a newly developed software tool CaverDock for screening databases of drugs against pharmacologically relevant targets. First, we evaluated the effect of rigid and flexible side chains on sets of substrates and inhibitors of seven different proteins. In order to assess the accuracy of our software, we compared the results obtained from CaverDock calculation with experimental data previously collected with heat shock protein 90α. Finally, we tested the virtual screening capabilities of CaverDock with a set of oncological and anti-inflammatory FDA-approved drugs with two molecular targets-cytochrome P450 17A1 and leukotriene A4 hydrolase/aminopeptidase. Calculation of rigid trajectories using four processors took on average 53 min per molecule with 90% successfully calculated cases. The screening identified functional tunnels based on the profile of potential energies of binding and unbinding trajectories. We concluded that CaverDock is a sufficiently fast, robust, and accurate tool for screening binding/unbinding processes of pharmacologically important targets with buried functional sites. The standalone version of CaverDock is available freely at https://loschmidt.chemi.muni.cz/caverdock/ and the web version at https://loschmidt.chemi.muni.cz/caverweb/.