Research in our laboratory is mostly oriented towards answering fundamental questions about the mechanism of action of various proteins that have biomedical and biotechnological importance. We explore protein structures and the interactions both within a protein and between the protein and molecules from the surrounding solvent, and how these interactions translate to a protein stability, dynamics and function. Regarding the protein function, we investigate mechanism responsible for: (i) facilitating transport of ligands to/from the functional sites that are deeply buried within the protein structures, (ii) recognition of cognate ligands by amino acid residues forming the functional sites of proteins, and (iii) in the case of enzymes, mechanism of biochemical conversion of bound ligands.
Research projects
Ligand transport pathways in proteins
At any given moment, living systems contain several thousand small organic molecules, both endogenous and exogenous, comprising the metabolome. To exert their function, the hosts of molecules need to arrive at their sites of action, mostly represented by protein surfaces or internal cavities. The transport of the metabolome is mainly governed by protein tunnels and channels. Such tunnels and channels secure the transport of ligands between different regions, and connect inner protein cavities with its surface, connect two or more different cavities, or connect even different cellular environments, such as in membrane proteins. The presence of very sophisticated transport processes markedly contributes to the symbiotic co-existence of individual chemical species within a single compartment or whole cell without the presence of overly disruptive interference.
Protein channels facilitate the regulated and very selective transport of ions and ligands across a membrane between different cellular compartments. Both tunnels and channels are often equipped with additional dynamical elements, called molecular gates, that can provide yet another level of control over transport processes. The role of channels in the function of various proteins has been the focus of intense research for years. Their importance is illustrated by the identification of many diseases caused by channel mutations. Such channel pathologies can severely impair the function of many physiological systems, manifested as various diseases, including epilepsy, hypertension, cystic fibrosis, diabetes, and cancer. To counter these malfunctions, many inhibitors or activators that affect transport through these channels have been identified.
Tunnels connect buried functional sites to the bulk solvent enabling the access of substrates and release of products. Many additional functions that are essential for the proper function of proteins exposed to interference from individual species that are present in the metabolome of the living cell are provided by tunnels: (i) enabling access of preferred substrates, while denying an access of non-preferred ones, (ii) avoiding the damage to the enzymes containing transition metals by their ligating, (iii) preventing damage to the cell by release of toxic intermediates to the proteins surrounding, (iv) enabling reactions that require the absence of water, and (v) temporal and spatial synchronization of reactions. Most enzymes likely possess tunnels. In fact, the presence of tunnels was already described for enzymes from six Enzyme Commission classes and four structural classes of proteins. Moreover, in many cases, tunnels are transient, meaning they cannot be readily identified from static crystal structures. Therefore, we can expect the discovery of tunnels in many other protein families. Recognizing the importance of transport processes for enzymatic catalysis, numerous protein engineering studies have successfully modified tunnels to improve enzymatic activity, specificity, enantioselectivity, and stability. Tunnels were established as important functional factors in enzyme catalysis relatively recently, but their role in cellular biochemistry and tunnel mutations in disease etiology has been largely overlooked. Although, many enzymes that are known to contain tunnels have been associated with the development of various ailments, including cancer, neurodegenerative disorders, autoimmune diseases, and inflammation. Inhibitors of some of these enzymes have been shown to bind to tunnels exclusively, thus confirming the proposed role of the tunnels in disease etiology and treatment.
To fill the gaps in our knowledge of ligand transport phenomena, our research focuses on answering the following questions:
- What are the structures, properties, and dynamics of tunnel networks in biologically relevant proteins?
- Which tunnels are traveled by particular ligands?
- How are relevant ligands transported through these tunnels?
- To what extent are tunnels influenced by their environment (e.g., solvent, small molecules, etc.)?
- What are the consequences of mutations of these tunnels?
Relevant literature:
Mandal N, Stevens JA, Poma AB, Surpeta B, Sequeiros-Borja C, Thirunavukarasu AS, Marrink SJ, Brezovsky J, 2026: Unlocking high-throughput investigation of transport tunnels in enzymes using coarse-grained simulation methods. Journal of Chemical Theory and Computation 22: 135-150. full text
Thirunavukarasu AS, Szleper K, Tanriver G, Marchlewski I, Mitusinska K, Gora A, Brezovsky J, 2025: Water migration through enzyme tunnels is sensitive to choice of explicit water model. Journal of Chemical Information and Modeling 65: 326–337. full text
Sethi A, Agrawal N, Brezovsky J, 2024: Impact of water models on the structure and dynamics of enzyme tunnels. Computational and Structural Biotechnology Journal 23: 3946-3954. full text
Mandal N, Surpeta B, Brezovsky J, 2024: Reinforcing Tunnel Network Exploration in Proteins using Gaussian Accelerated Molecular Dynamics. Journal of Chemical Information and Modeling 64: 6623-6635. full text
Sarkar DK, Surpeta B, Brezovsky J, 2024: Incorporating prior knowledge in the seeds of adaptive sampling molecular dynamics simulations of ligand transport in enzymes with buried active sites. Journal of Chemical Theory and Computation 20: 5807-5819. full text
Sequeiros-Borja C, Bartlomiej Surpeta, Thirunavukarasu AS, Dongmo Foumthuim CJ, Marchlewski I, Brezovsky J, 2024: Water will find its way: transport through narrow tunnels in hydrolases. Journal of Chemical Information and Modeling 64: 6014-6025. full text
Sequeiros-Borja C, Surpeta B, Marchlewski I, Brezovsky J, 2023: Divide-and-conquer approach to study protein tunnels in long molecular dynamics simulations. MethodsX 10C: 101968. full text
Brezovsky J, Thirunavukarasu AS, Surpeta B, Sequeiros-Borja CE, Mandal N, Sarkar DK, Dongmo Foumthuim CJ, Agrawal N, 2022: TransportTools: a library for high-throughput analyses of internal voids in biomolecules and ligand transport through them. Bioinformatics 38: 1752-1753. full text
Brezovsky J, Babkova P, Degtjarik O, Fortova A, Gora A, Iermak I, Rezacova P, Dvorak P, Kuta Smatanova I, Prokop Z, Chaloupkova R, Damborsky J, 2016: Engineering a De Novo Transport Tunnel. ACS Catalysis 6: 7597-7610. full text
Gora A, Brezovsky J, Damborsky J, 2013: Gates of Enzymes. Chemical Reviews 113: 5871–5923. full text
Marques SM, Daniel L, Buryska T, Prokop Z, Brezovsky J, Damborsky J, 2016: Enzyme Tunnels and Gates as Relevant Targets in Drug Design. Medicinal Research Reviews (in press, doi:10.1002/med.21430). full text
Brezovsky J, Chovancova E, Gora A, Pavelka A, Biedermannova L, Damborsky J, 2013: Software Tools for Identification, Visualization and Analysis of Protein Tunnels and Channels. Biotechnology Advances 31: 38-49. full text
Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, Khabiri M, Plevaka M, Kulik D, Kuta Smatanova I, Rezacova P, Ettrich R, Bornscheuer UT, Damborsky J, 2013: Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel. Angewandte Chemie International Edition 52: 1959-1963. full text
Marques SM, Brezovsky J, Damborsky J, 2016: Role of Tunnels and Gates in Enzymatic Catalysis. In: Svendsen, A., Understanding Enzymes: Function, Design, Engineering, and Analysis, Pan Stanford Publishing, pp. 421-463. full text
Prokop Z, Gora A, Brezovsky J, Chaloupkova R, Stepankova V, Damborsky J, 2012: Engineering of Protein Tunnels: Keyhole-lock-key Model for Catalysis by the Enzymes with Buried Active Sites. In: Lutz, S., Bornscheuer, U.T. (Eds.), Protein Engineering Handbook, Wiley-VCH, Weinheim, pp. 421-464. full text
Understanding the effects of mutations
Frequently, native structures of proteins become modified to a varying extent as a consequence of mutations. These arise either from naturally occurring processes or from acts of protein engineers aiming to alter a protein's properties using molecular biology methods. The ability to predict the effects of such mutations is essential for precision/personalized medicine to pinpoint those mutations likely associated with the development of various diseases for detailed investigation. Conversely, it is often preferable to avoid predicted harmful mutations during the design of a modified protein for experimental protein engineering/construction.
To uncover the effects of particular mutations, we perform bioinformatic analysis and molecular simulations to compare structure-dynamics-function relationships of mutant and native proteins. Then we use the acquired knowledge to rationally design mutants with improved properties of interest and to develop computational tools for automated predictions of mutations' effects.
Relevant literature:
Pakuła K, Sequeiros-Borja C, Biała-Leonhard W, Pawela A, Banasiak J, Bailly A, Radom M, Geisler M, Brezovsky J, Jasiński M, 2023: Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters. Cellular and Molecular Life Sciences 80: 105. full text
Grulich M, Brezovsky J, Stepanek V, Palyzova A, Maresova H, Zahradnik J, Kyslikova E, Kyslik P, 2016: In-silico Driven Engineering of Enantioselectivity of a Penicillin G Acylase towards Active Pharmaceutical Ingredients. Journal of Molecular Catalysis B: Enzymatic (in press, doi:10.1016/j.molcatb.2016.11.014). full text
Bendl J, Stourac J, Salanda O, Pavelka A, Wieben ED, Zendulka J, Brezovsky J, Damborsky J, 2014: PredictSNP: Robust and Accurate Consensus Classifier for Prediction of Disease-Related Mutations. PLoS Computational Biology 10: e1003440. full text
Bendl, J., Musil, M., Stourac, J., Zendulka, J., Damborsky, J., Brezovsky, J., 2016: PredictSNP2: A Unified Platform for Accurately Evaluating SNP Effects by Exploiting the Different Characteristics of Variants in Distinct Genomic Regions. PLoS Computational Biology 12: e1004962. full text
Bendl J, Stourac J, Sebestova E, Vavra O, Musik M, Brezovsky J, Damborsky J, 2016: HotSpot Wizard 2: Automated Design of Site-Specific Mutations and Smart Libraries in Protein Engineering. Nucleic Acids Research 44: W479-487. full text
Bednar D, Beerens K, Sebestova E, Bendl J, Khare S, Chaloupkova R, Prokop Z, Brezovsky J, Baker D, Damborsky J, 2015: FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants. PLoS Computational Biology 11: e1004556. full text
Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, Khabiri M, Plevaka M, Kulik D, Kuta Smatanova I, Rezacova P, Ettrich R, Bornscheuer UT, Damborsky J, 2013: Engineering Enzyme Stability and Resistance to an Organic Cosolvent by Modification of Residues in the Access Tunnel. Angewandte Chemie International Edition 52: 1959-1963. full text
Discovery of bioactive ligands
Specific and rather tight binding of small organic molecules (ligands) by an enzyme represents the first prerequisite for a ligand to become an enzyme's substrate if it binds in an appropriate binding mode and is susceptible to catalysis, or an inhibitor if the ligand's binding interferes with the reaction mechanism.
Traditionally, identification of novel active ligands has been achieved through expensive, time-consuming, and often ineffective biochemical assays. Fortunately, the process could be made more effective by combining high-throughput robotic screening and computational virtual screening.
To identify diverse lead ligands for follow-up experimental verification, QSAR studies, or more focused screening, we perform virtual screenings based on the structures and mechanisms of action of specific proteins or on known active ligands.
Relevant literature:
Nikulenkov F, Carbain B, Biswas R, Havel S, Prochazkova J, Sisakova A, Zacpalova M, Chavdarova M, Marini V, Vsiansky V, Weisova V, Slavikova K, Biradar D, Khirsariya P, Vitek M, Sedlak D, Bartunek P, Daniel L, Brezovsky J, Damborsky J, Paruch K, Krejci L, 2025: Discovery of new inhibitors of nuclease MRE11. European Journal of Medicinal Chemistry 285: 117226. full text
Daniel L, Buryska T, Prokop Z, Damborsky J, Brezovsky J, 2015: Mechanism-Based Discovery of Novel Substrates of Haloalkane Dehalogenases using in Silico Screening. Journal of Chemical Information and Modeling 55: 54-62. full text
Grulich M, Brezovsky J, Stepanek V, Palyzova A, Kyslikova E, Kyslik P, 2015: Resolution of α/β-Amino Acids by Enantioselective Penicillin G Acylase from Achromobacter sp. Journal of Molecular Catalysis B: Enzymatic 122: 240-247. full text
Buryska T, Daniel L, Kunka A, Brezovsky J, Damborsky J, Prokop Z, 2016: Discovery of Novel Haloalkane Dehalogenase Inhibitors. Applied and Environmental Microbiology 82: 1958-1965. full text