Research at our laboratory is mostly oriented towards answering fundamental questions concerning the mechanism of action of various proteins of 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 moment, living systems contain several thousands of small organic molecules both endogenous and exogenous, 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. They secure the transport of ligands between different regions, connecting inner protein cavities with its surface, two or more different cavities, or even different cellular environments such as in the membrane proteins. The presence of very sophisticated transport regulation markedly contributes to symbiotic co-existence of individual chemical species within a single compartment or whole cell without the presence of overly disruptive interferences.

Protein channels facilitate regulated and very selective transport of ions and ligands across a membrane between different cell 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 identification of many diseases caused by channel mutations. Those channel pathologies were implicated to severely impair the function of many physiological systems manifesting into a variety of diseases like epilepsy, hypertension, cystic fibrosis, diabetes, and cancer. To counter these malfunctions, many inhibitors or activators able to affect the transport through those channels were identified.

The tunnels connect buried functional sites to the bulk solvent enabling access of substrates and release of products. Many additional functions essential for the proper function of proteins exposed to the interference of individual species 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 of the enzymes are likely to 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, the tunnels are transient, which means they cannot be readily identified from static crystal structures. Therefore, we may yet expect the discovery of tunnels in many other protein families. Recognizing the importance of transport processes to enzymatic catalysis, the number of protein engineering studies successfully modified tunnel to improve enzyme activity, specificity, enantioselectivity, and stability. Since it has been only recently that the tunnels were established as important functional factors in the enzyme catalysis, the roles of tunnels in the cellular biochemistry and tunnel mutations in the disease development has been largely overlooked. However, many enzymes known to contain tunnels were already connected with a development of various ailments like cancer, neurodegenerative disorders, autoimmune diseases or inflammation. For some of them, inhibitors were identified that bind to the tunnels exclusively, confirming the proposed role of the tunnels in the development of diseases and their treatment.

To help filling the existing gaps in our knowledge on ligand transport phenomena, our research has been designed to answer following questions:

  1. What are the structure, properties, and dynamics of tunnel networks in biologically relevant proteins?
  2. Which tunnels are traveled by particular ligands?
  3. How are the relevant ligands transported through these tunnels?
  4. To what extent are tunnels influenced by their environment (solvent, small molecules, etc)?
  5. What are the consequences of mutations occurring in these tunnels?

Relevant literature:

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 various extent as a consequence of mutations. These arise either due to naturally occurring processes or due to acts of protein engineers aiming to alter protein's properties by molecular biology methods. The ability to predict the effect of such mutations is essential in precision/personalized medicine to pinpoint those mutations that are likely associated with the development of various diseases for detailed investigation. Conversely, it is often favorable to avoid those mutations that are predicted as harmful during the design of 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:

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 enzyme represents the first prerequisite for such ligand to become an enzyme's substrate if it binds in appropriate binding mode and is susceptible to catalyzed reaction, or an inhibitor if the binding of the ligand interferes with the reaction mechanism.

Traditionally, identification of novel active ligands has been achieved by performing expensive, time-consuming and often rather ineffective biochemical assays. Fortunately, the process could be made more effective by using coupled high-throughput-robotic screenings and computational virtual screenings.

To identify diverse lead ligands for follow-up experimental verification, QSAR studies or more focused screening, we perform virtual screenings based on either on structure and mechanism of action of specific proteins or already known active ligands.

Relevant literature:

Damborsky J, Nikulenkov F, Sisakova A, Havel S, Krejci L, Carbain B, Brezovsky J, Daniel L, Paruch K, 2015: Pyrazolotriazines as Inhibitors of Nucleases. Masaryk University, Brno, Czech Republic. Patent WO 2015/192817 A1. 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