Our group is specialized in structural biology techniques, such as X-ray crystallography, SAXS and NMR, in combination with biochemical and molecular biology essays, and computer modelling, to study structure-functional relationships of various proteins. Our objects of interest include proteins used in biotechnological applications; bacterial and human proteins that are potential targets for drug treatment; and proteins from various organisms, that are important for understanding of biological processes. The main projects are described below.
Halogenated compounds represent one of the largest groups of environmental pollutants, that historically were used as agricultural pesticides, organic solvents, or as intermediates for the synthesis of certain chemicals. As a number of halogenated compound exhibit neurotoxic and carcinogenic effects, and are poorly biodegradable, they were banned from use under the Stockholm Convention on persistent organic pollutants. Haloalkane dehalogenases (EC 220.127.116.11) are responsible for one of the key reactions in the pathway of aerobic mineralization of halogenated compounds. They catalyze the cleavage of the carbon-halogen bond in halogenated aliphatic pollutants, resulting in formation of a corresponding alcohol, a halide ion and a proton. Therefore, these enzymes have applications in biosensing and bioremediation of halogenated-compounds-pollution, and also in biosynthesis, cellular imaging and protein immobilization. Structural analysis of catalytic mechanism is an important step in rational design of haloalkane dehalogenases for biotechnlogical applications.
Bacterial haloacid dehalogenases (HADs) superfamily includes phosphoesterases, ATPases, phosphonatases, dehalogenases, and sugar phosphomutases with highly diverse substrate specificity. The enzymes from this superfamily are found in the organisms from all three superkingdoms of life, displaying numerous very disparate biological functions; and a variety of hydrolytic enzyme activities remain to be described. Many of the proteins with known enzymatic activities in the HAD superfamily are involved in detoxification of xenobiotics or metabolic by-products, displaying potential for biotechnological applications. HADs are able to cleave C-Cl, P-C, or P-O bonds in various substrates using conserved nucleophilic aspartic acid in the active site. Apart from adapting these enzymes for industrial applications, analysis of HAD structures will help to understand how these enzymes evolved to act on a wide range of substrates. Also, structural information should facilitate the prediction of important functional residues or regions in members of the superfamily with unknown function.
Glyceraldehyde dehydrogenase and dihydroxyacid dehydratase
Currently cell-free approaches have practical applications in cellular metabolic pathways investigation, protein and peptide synthesis and evolution, small molecule production, and non-natural product synthesis. These techniques are environmentally gentle compared to chemical synthesis using natural fossil resources, and display high flexibility for rational programming of biosynthetic networks. Recently, a completely artificial minimized reaction cascade was constructed for the conversion of glucose to ethanol or isobutanol using single cofactor NAD+, which is completely recycled within the cascade. The two key enzymes of this pathway are glyceraldehyde dehydrogenase (AlDH) and dihydroxyacid dehydratase (DHAD). To be implemented in the cascade AlDH needs to have a very high substrate specificity, DHAD, on a contrary, catalyzes several reactions within the cascade and requires broad substrate specificity. The enhancement of the enzymes requires screening potential candidates for optimal catalytic properties with further structure-based rational engineering.
Photosystem II from higher plants (PSII)
Photosystem II (PSII) is a multiprotein-pigment complex embedded in the thylakoid membrane of plants, algae, and cyanobacteria that catalyzes the light-driven oxidation of water to molecular oxygen. Additionally, PSII regulates (light) energy distribution, acts as a stress sensor and is a target for herbicides. It is subdivided into two functional domains: (1) the electron transfer domain and (2) the oxygen-evolving complex (OEC), consisting of more than 20 different protein subunits and beyond 50 pigment co-factors and several ions. Structural data on proteins fold, protein-protein and protein-cofactor interactions in the PSII complex will enable one to elucidate the details of functioning, molecular mechanisms of regulation and signal transduction. A number of full-complex structures as well as single proteins were determined for PSII from cyanobacteria and algae. Only one structure of the PSII supercomplex from higher plants is currently available, namely, from spinach (Spinacia oleracea). Individual proteins of the complex were also structurally characterized for Pisum sativum and Arabidopsis thaliana. However, a lot of questions regarding the details of PSII functioning remain unanswered and require structural information about complexes from other organisms, preferably, at higher resolution.
Fe-regulated proteins from Neisseria meningitidis
Neisseria meningitidis is found in nasopharynges and oropharynges in about 10% of population without causing any symptoms. In small fraction of hosts, however, meningococci invade the bloodstream and can cross the blood-brain barrier, causing septicemia and/or meningitis with high mortality rates. Under conditions of iron deficiency N. meningitides produces so-called Fe-regulated proteins (Frp). These proteins have the characteristic carboxy-proximal glycine and aspartate-rich RTX (repeat-in-toxin) motifs, which are repetitions of a nonapeptide L-X-G-G-X-G-(D/N)-D-X. Many RTX proteins are known to serve as virulence factors for Gram-negative pathogens such as genera Escherichia, Proteus, Actinobacillus, Morganella, Pasteurella, Bordetella, and Vibrio. However, for some of these proteins (e.g. FrpC) no cytotoxic activity was detected, and their biological function remains unknown. Study of FrpC-like proteins will help to uncover further details of proteins functions and, potentially, indicate new targets for medical treatment.
Bordetella pertussis is highly specific to human bacterial pathogen, it causes whooping cough, a severe respiratory tract infection, in infants and children, and also infects adults. Bordetella exploits an immunosuppressive cytokine, interleukin-10 (IL-10), to evade the host immune system. Effectors (e.g. BopN) are transported into the host cell via the type III secretion system, where it induces enhanced production of IL-10. Also, the BopN effector translocates itself into the nucleus and is involved in the down-regulation of mitogen-activated protein kinases. Involvement of BopN in B. pertussis stealth strategy to shut off host immune reaction makes it an interesting target for putative vaccines. Currently, there is no structure of BopN or homologous protein available to provide necessary information for structure-driven drug design.
The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol, the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. The cyclization of 2,3-oxidosqualene is catalyzed by oxidosqualene cyclases (OSCs or triterpene synthases), and leads to the formation of either sterol or triterpene scaffolds, in a complex series of concerted reaction steps. 2,3-oxidosqualene is a precursor of various triterpenes, which are present in plant surfaces; and of cholesterol in mammals.
The majority of the so-called carrier proteins (CP) is known for their ability to bind, store, and transport heme. Furthermore, tick carrier proteins are presumable transporters of lipids and proteins. The antioxidation activity is expected from CPs due to the binding of heme. The CP of the tick Dermacentor marginatus, hemelipoglycoprotein (HLGP), is one of the most abundant proteins of D. marginatus hemolymph. Its expression varies among tick species and depends also on developmental stage and blood-feeding behavior. However, in general, CPs are non-sex-linked and are present in unfed ticks as well. Although HLGP can be used by ticks to obtain iron from hosts, the ability to sequester heme is probably not the only important function of these proteins.