Antiviral Defence Systems in Bacteria
Phages are the most abundant organisms in the biosphere and the major parasites of bacteria. They infect bacteria in order to replicate and usually kill bacteria when the replication is completed. In response to the phage threat, bacteria developed multiple defence barriers for countering and fighting viral attacks. In the Department of Protein-Nucleic Acids Interactions we aim to understand the structure-function relationships of enzymes and enzyme assemblies that contribute to the bacteria defence systems that target invading nucleic acids. We are particularly interested in the molecular machinery involved in the CRISPR-Cas function and the structural and molecular mechanisms of other antiviral defence systems including prokaryotic Argonautes, BREX, toxin-antitoxin systems and others. We are using X-ray crystallography, mutagenesis and functional biochemical as well as biophysical assays to acquire more information on these systems.
CRISPR-Cas has been recently discovered as a prokaryotic antiviral defence system that hijacks short fragments of invasive DNA as spacers and subsequently uses them as templates to generate specific small RNA molecules that combine with Cas proteins into effector complexes that trigger the degradation of foreign nucleic acid. In this respect, CRISPR-Cas systems constitute an adaptive microbial immune system that provides an acquired resistance against invaders. CRISPR systems are very diverse, and we aim to understand the molecular and structural mechanisms of immunity provided by different CRISPR-Cas systems.
In recent years, we have focused on different aspects of CRISPR-Cas systems, in collaboration with Dr. D. Wigley (Imperial College London), Dr. R. Seidel (Universität Leipzig), Dr. M. D. Szczelkun (Bristol University), Dr. J. Young (Corteva), Dr. C. Venclovas (Vilnius University), Dr. M. Bochtler (IUCMB), Drs. K. Makarova and E. Koonin (NIH). We continue to explore molecular mechanisms behind cyclic oligoadenylate signalling pathway discovered by us in 2017 and other proteins related to CRISPR-Cas or other antiviral defence systems.
- Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G., Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science. 2017, 357: 605–
- Sasnauskas G, Siksnys V. CRISPR adaptation from a structural perspective. Curr Opin Struct Biol. 2020, 19, 65: 17–
- Makarova, K. S., Timinskas, A., Wolf, Y. I., Gussow, A. B., Siksnys, V., Venclovas, Č., Koonin, E. V. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic Acids Res. 2020, 48(16): 8828– doi: 10.1093/nar/gkaa635.
- Songailiene, I., Rutkauskas, M., Sinkunas, T., Manakova, E., Wittig, S., Schmidt, C., Siksnys, V., Seidel, R. Decision-making in cascade complexes harboring crRNAs of altered length. Cell Rep. 2019, 28(12): 3157–e4. doi: 10.1016/j.celrep.2019.08.033.
- Wilkinson, M., Drabavicius, G., Silanskas, A., Gasiunas, G., Siksnys, V., Wigley, D. B. Structure of the DNA-bound spacer capture complex of a type II CRISPR-Cas system. Mol Cell. 2019, 75(1): 90–101.e5. doi: 10.1016/j.molcel.2019.04.020.
Novel Toxin-Antitoxin System Related to I-D CRISPR-Cas System
HEPN-MNT toxin-antitoxin (TA) system is encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. Using biochemical and structural methods, we showed that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. We propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin. The I-D CRISPR-Cas system present in A. flos-aquae contains a putative Cas3-like ATPase-helicase; thus, the HEPN-MNT TA system could become activated due to additional ATP degradation by CRISPR-Cas3 in response to phage infection. In this case, I-D CRISPR-Cas ATPase-controlled activation of HEPN RNase in A. flos-aquae would be analogous to activation of the auxiliary Csm6 RNase by cyclic oligoadenylate produced by the type III CRISPR-Cas system in response to phage infection. However, the exact mechanism of the HEPN-MNT system action and its possible crosstalk with the CRISPR-Cas system remain to be established (Songailiene et al. Mol. Cell. 2020, S1097-2765(20)30834-0).
Crystal structure of di_AMPylated HEPN ribonuclease
Proposed mechanism of action of A. flos-aquae HEPN-MNT toxin-antitoxin system
Studies of 5′ Modifications to CRISPR–Cas9 Guide RNA
A key aim in exploiting CRISPR–Cas is guide RNA (gRNA) engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design (Mullally et al., Nucleic Acids Res. 2020, 48, 6811–6823).
gRNA heatmap showing gRNA regions which tolerate (purple), partially tolerate (orange) and do not tolerate (grey) modification
Bioelectrochemical Systems in Biosensors and Bioreactors
Bioelectrochemical Systems in Biosensors and Bioreactors
Mediated and direct electron transfer (ET) coupling of enzymes to electrodes is important in realizing bioelectrocatalysis, which is often exploited as a basic principal of biosensors, biofuel cells, and other bio-based devices. These technologies exploit the inherent enzyme substrate specificity, for example, enzyme-based biosensors excel in direct measurement of single compound in presence of interfering materials in complex media such as blood. On the other hand, if the power density generated by enzyme-based electrode is high enough, biofuel cells can be constructed, where bioelectrodes selectively oxidise and reduce abundant fuel (i.e. glucose and oxygen) and provide electric power for implantable devices. The fragile nature of proteins dictates that the electrochemical properties of such biodevices degrade over time. Therefore, a number of techniques are developed to protect the biomolecule and extend the working period of device. The shortcoming could be avoided whatsoever by adsorbing live, whole cells on electrodes at the expense of reduced power density.
Our team is proficient at constructing bioelectrochemical systems by wiring oxidoreductases to gold and carbon based electrode surfaces [1–3]. Our team is also developing bioreactor systems, where wasteful saccharide substrates are selectively oxidised and high-value oxidation products are produced. For such an approach, we utilize bi-enzymatic reaction with biosensor-based microprocessor-controlled substrate dispensing. In order to obtain a self-regulating system, the fluid dispensing and sensor devices are coordinated by an advanced algorithm embedded in microcontroller-based electronic system. All the custom components were designed and produced by our team. Recently, molecularly imprinted polymers based on polypyrrole and polyaniline preparations have intensively been studied for sensor electrode application ; the approach should help in finding new ways to discover new supramolecular systems for small biomolecule detection.
1. Ratautas, D., Dagys, M. Nanocatalysts containing direct electron transfer-capable oxidoreductases: recent advances and applications. Catalysts. 2020, 10: 9.
2. Gineitytė, J., Meškys, R., Dagys, M., Ratautas, D. Highly efficient direct electron transfer bioanode containing glucose dehydrogenase operating in human blood. J. Power Sources. 2019, 441: 227163.
3. Dagys, M., Laurynėnas, A., Ratautas, D. et al. Oxygen electroreduction catalysed by laccase wired to gold nanoparticle via the trinuclear copper cluster. Energy & Environmental Science. 2017, 10: 498.
4. Bagdžiūnas, G. Theoretical design of molecularly imprinted polymers based on polyaniline and polypyrrole for detection of tryptophan. Mol. Syst. Des. Eng. 2020, 5: 1504.
Dehydrogenases in Custom Sensor-Controlled Bioreactors
The use of the dehydrogenases to oxidize substrates in bioreactors is very attractive due to their broad substrate specificity and high catalytic activity. However, the application requires an effective mediated enzyme reoxidation method. The mediator in regeneration scheme must be highly reactive with the enzyme to regenerate; all forms of the mediator must be stable, nontoxic and cheap. The oxidized mediator form is produced in reaction with heme peroxidase, which exhibits high catalytic activity at pH 7.0 and broad substrate specificity. Peroxidase uses hydrogen peroxide as co-substrate to oxidase mediators. To avoid the hydrogen peroxide-induced enzyme inactivation, the addition of hydrogen peroxide to the reactor mixture was performed in very small doses of 40 nL by using the syringe pump developed by our team. The rate of dosage was controlled by analysing the data of our custom highly-sensitive hydrogen peroxide and optical, oxidized mediator-form sensors, all combined into microcontroller-coordinated control algorithm. As of today, the turnover number of PQQ glucose dehydrogenase in model reactor reaches ~1x107, which means one can produce a valuable product at ~0,65 Eur/g and expect to sell it for about a hundred times more (RCL grant No. 01.2.2-LMT-K-718-01-0019).
Theoretical Design of Molecularly Imprinted Polymers for Analysis
Creation of molecularly imprinted polymers (MIPs) as the supramolecular systems with tailor-made binding sites complementary to template molecules in shape, size and functional groups is an important task for the analytical, physical and theoretical chemistries. In this work, the polypyrrole and polyaniline-based host - guest MIPs were theoretically studied for the detection of tryptophan. These simulations showed that polyaniline is not suitable for the selective detection of tryptophan due to high flexibility of its chains and low energy of intermolecular interactions. In contrast, the polypyrrole-based hosts can be used to detect tryptophan, because all these simulated forms shaped the bow-shaped inner cavity with the strongly coordinated target molecule. Moreover, the insights will help in finding new ways to discover new supramolecular systems for small biomolecule detection  (RCL grant No. S-MIP-20-45).
Self-Organization of Bacteria
Bioanalytical systems can be constructed by using whole-cell biosensors, where bacteria are grown on electrode surfaces. We use bioluminescence imaging to record images of liquid mixed cultures of the lux-gene reporter E. coli and other bacteria in microtiter plate wells and in vertical Hele-Shaw cells. Analysis of the experimental data together with mathematical modelling suggests the following interpretation of pattern formation (right figure: a) and b) show typical side-view and top-view images of cylindrical samples (bar – 1 mm), the scheme of a system that forms spatiotemporal patterns is shown in c). The evaporation- and settling-driven instability of the surface layer results in formation of oxygenated plumes. In the vicinity of the plumes, active cells (grey circles) ‘aggregate’ and ‘grow’ at the expense of passive cells (white circles). These studies were partly funded by RCL grant No. S-MIP-17-98.
Biological Modification of DNA and RNA
Biological Modification of DNA and RNA
Epigenetic Modifications of DNA and RNA in Mammals
In recent years, epigenetic phenomena have become a major focus in studies of embryonic development, genomic imprinting and complex human diseases. One of the best-understood epigenetic mechanisms is enzymatic DNA methylation. In the mammalian genome, cytosines in CpG dinucleotides are often methylated to 5-methylcytosine (m5C), which is brought about by combined action of three known AdoMet–dependent DNA methyltransferases (DNMTs). DNA methylation profiles are highly variable across different genetic loci, cell types and organisms, and are dependent on age, sex, diet and disease. Besides m5C, certain genomic DNAs contain detectable amounts of 5-hydroxymethylcytosine (hmC) and lower levels of 5-formylcytosine and 5-carboxylcytosine (caC), which are produced by the oxidation of m5C residues by TET oxygenases. However, many details of how these modifications are established at specific loci and how they control cellular events remain obscure .
More than 160 chemically distinct covalent modifications have been detected across various RNA species in prokaryotic and eukaryotic cells. One of the most abundant and important RNA modifications is methylation of the 2'OH group. miRNAs, piRNAs and siRNAs are small non-coding RNA molecules that control gene activity in a homology-dependent manner. Biogenesis of miRNAs and siRNAs in plants involves a methylation step catalysed by the HEN1 methyltransferase, whereas piRNAs are similarly modified in animals [2,3].
Following our long-standing interest in mechanistic studies of DNA MTases, we turned our focus on advancing DNA and RNA modification analysis and its applications for studies of epigenetic mechanisms [3,4]. Our current ERC-supported studies seek to gain in-depth understanding of how the DNA methylation patterns are established by the three known DNMTs during differentiation and development. Here, our efforts are devoted to devising single-cell methodologies that permit precise determination of where and when the methylation marks are deposited by the individual DNMTs inside living cells (see Figure above).
1. Liutkevičiūtė, Z., Kriukienė, E. Ličytė, J., Rudytė, M., Urbanavičiūtė, G., Klimašauskas, S. Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases. J. Am. Chem. Soc. 2014, 136: 5884–5887.
2. Baranauskė, S., Mickutė, M., Plotnikova, A., Finke, A., Venclovas, Č., Klimašauskas, S., Vilkaitis, G. Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins. Nucleic Acids Res. 2015, 43(5): 2802–2812.
3. Mickutė, M., Nainytė, M., Vasiliauskaitė, L., Plotnikova, A., Masevičius, V., Klimašauskas, S., Vilkaitis, G. Animal Hen1 2'-O-methyltransferases as tools for 3'-terminal functionalization and labelling of single-stranded RNAs. Nucleic Acids Res. 2018, 46(17): e104.
4. Kweon, S. M., Chen, Y., Moon, E., Kvederavičiūtė, K., Klimašauskas, S., Feldman, D. E. An adversarial DNA N6-methyladenine-sensor network preserves polycomb silencing. Mol. Cell. 2019, 74: 1138–1147.
5. Tomkuvienė, M., Mickutė, M., Vilkaitis, G., Klimašauskas S. Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA. Curr. Opin. Biotechnol. 2019, 55: 114–123.
Enzymatic Hydroxylation and Excision of Extended 5-Methylcytosine Analogues
Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multi-step demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated carbon-carbon bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjacent to an sp1 or sp2 system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions (Tomkuvienė et al. J. Mol. Biol. 2020, 423: 6157–6167).
Photocage-Selective Capture and Light-Controlled Release of Target Proteins
Photochemical transformations enable exquisite spatio-temporal control over biochemical processes, however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. We went on to create a high affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically-encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues which were used for raising a high affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a series of DMNB-caged model proteins and DNA cytosine-5 methyltransferase enzymes from complex biological mixtures. The proposed orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays (Rakauskaitė et al. iScience. 2020, 23(12): 101833; Klimašauskas et al., LT2020539).
Hen1 Methyltransferase-Directed RNA Capture and Sequencing of miRNAs and Bacterial Small RNAs in Probiotic Lactobacillus casei
Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualization, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3’ adapter ligation in strand-specific RNA sequencing, remain challenging despite numerous optimizations. Here we remedy this limitation by adapting two small RNA 2’-O-methyltransferases, ssRNA-specific DmHen1 and dsRNA modifying AtHEN1, for orthogonal chemo-enzymatic click tethering of a 3’ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. We show by profiling a reference miRNA pool and the small RNA transcriptome of probiotic Lactobacillus casei BL23 that the developed methyltransferase-captured 3’ RNA sequencing technique, meCap-seq, can advance analysis of eukaryotic and prokaryotic ssRNA pools. Our findings provide a valuable resource for studies of the regulatory RNome in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells (EP3271478 B1; Mickutė et al., submitted).
Electrochemical biosensors for real-world applications
Electrochemical Biosensors for Real-World Applications
Biosensors are handy devices, which can rapidly detect and measure a variety of specific compounds. Those devices can significantly improve the quality of life for patients suffering from a variety of diseases by helping the medical personnel to diagnose diseases faster and more accurately as well as help to evaluate other significant factors. However, to develop a biosensor operating with adequate performance for real-world applications is a tedious task. Real media samples are complex, making an accurate detection difficult. For example, human blood, the most clinically relevant sample, is composed of thousands of different compounds with unique properties, a variety of blood cells and countless number of proteins. To discern a single type molecule from this entire composition, a biosensor should be specifically designed and engineered. Typically, the surface of the electrochemical biosensor is covered with compound-specific enzyme, unique membranes are designed to reject the interfering compounds, complex electronics and mathematical analysis models are used to increase the signal-to-noise ratio. Our department designs such biosensors capable of analysing various peculiar media obtained from nature. The significance of some of analytes we are interested in are not yet realized, e.g. glutamate concentration in mice brain media or release of glucose as a stress factor in fish tanks.
Our department has been working with the development of biosensors for real-world applications involving devices for clinical practice, accumulating sizeable competencies in this field. In recent works, we have demonstrated a new type ofnanomaterial-based glucose biosensors, which could operate with high precision and accuracy in clinically-relevant fluids – human serum  and blood . This type of biosensors could be miniaturized and implanted in patients’ bodies and be used for real-time monitoring of glucose. Additionally, we have developed biosensors for low-level glucose measurements and demonstrated that those biosensors could be used for monitoring the stress level of juvenile trout fish . Clinically relevant projects in our department are carried out in cooperation with Vilnius University Hospital Santaros Klinikos and are related to the diagnosis and prognosis of the clinical outcome of patients undergoing renal replacement therapy as well as of those with other specific conditions, e.g. acute pancreatitis .
1. Ratautas, D.et al. Real-time glucose monitoring system containing enzymatic sensor and enzymatic reference electrodes. Biosensors & Bioelectronics. 2020, 164: 112338.
2. Makaras, T., Razumienė, J. et al. A new approach of stress evaluation in fish using β-d-Glucose measurement in fish holding-water. Ecological Indicators. 2020, 109: 105829.
3. Gineitytė, J. et al. Highly efficient direct electron transfer bioanode containing glucose dehydrogenase operating in human blood. Journal of Power Sources. 2019, 441: 227163.
4. Razumienė, J. et al. The synergy of thermally reduced graphene oxide in amperometric urea biosensor: application for medical technologies. Sensors. 2020, 20: 4496.
Amino Acids’ Biosensor in Clinical Applications
We are developing an electrochemical biosensor platform for the fast, accurate and cheap detection as well as quantification of total and specific (glutamate, glutamine, lysine) amino acids present in biologically relevant fluids – dialysate buffer after haemodialysis and human serum/blood. The analysis of the amino acids in biological fluids for clinical applications is an unresolved challenge worldwide, since the concentration levels of amino acids are very low (typically 1–100 µmol/l or less) and create challenges. The major applicable methods for amino acid analysis in clinical diagnostics involve either commercial colorimetric (e.g., ELISA) kits or chromatographic amino acid analysers, which are costly, time-consuming and require highly trained scientific personnel to operate. For such reasons the analysis of amino acid in hospitals for clinical diagnostics and monitoring is not a usual routine procedure. Consequently, the extreme diagnostic potential of amino acids present in biological fluids is not fully utilized and may be overlooked (RCL grant No. 01.2.2-LMT-K-718-03-0005 and S-EJPRD-20-1).
Biosensors for Fish Stress Level Control
Recently, our long-lasting glucose biosensor technology has been upgraded for measuring nanomolar concentrations of glucose in fish tanks. This has been proven statistically relevant in determining levels of stress experienced by rainbow trout juveniles . The developed biosensor has several advantages over conventional methods, i.e. a wide linear range, high sensitivity, good selectivity, long-term stability and ability to act in non-pre-treated turbid media. In future, glucose measurement in water using an appropriate biosensor could be a useful tool for assessing environmental risk for assessing different contaminant exposure and effects (RCL grant No. 09.3.3-LMT-K-712-19-0110).
Urea Biosensors with Thermally Reduced Graphene Oxide
We discovered that the synergy of the electrode based on thermally reduced graphene oxide (TRGO) nanoparticles in combination with urease allowed the development of a promising urea biosensor for clinical trials. According to the data of recently reported (2015–2019) urea biosensors, the features of TRGO-based urea biosensor include advanced analytical characteristics such as good sensitivity, wide linear range, long storage and operational stability for the accomplishment of more than 300 samples of clinical trials without significant change. Low-cost design, good reproducibility and fast response time allowed applying the biosensor for monitoring of urea levels in real samples such as urine, blood and dialysate collected during the haemodialysis (HD) procedure. The accuracy of the biosensor action was validated by approved methods on a base of a large number of measurements.
The experiments confirmed that urea measurement in urine and in spent dialysate possess a great potential as a tool for evaluation of dialysis adequacy as well as a step leading to point-of-care non-invasive technologies (RCL grant No. 01.2.2-LMT-K-718-01-0025).
Crystallography and Molecular modelling
Crystallography and Molecular Modelling
Modelling matter at atomic level is important for structural biology, material science, physics and (bio)chemistry. These methods become increasingly important with the growth of available computing power, availability of large amounts of high quality, machine-readable computer data and advent of new methods such as machine learning. Our approach to molecular modelling consists of organizing available data into well-defined, curated machine-readable open databases, and then using these databases for scientific inferences applying thoroughly documented, reproducible computation procedures.
The main collection of data that we maintain is the Crystallography Open Database (COD). Over 15 years of development, the COD supervised by the international Advisory Board (of which S. Gražulis and. A. Merkys are members) was transformed into the world’s largest open access small molecule crystal data collection. Containing currently close to half a million records, the COD is widely used by researchers worldwide (the two seminal publications together attracted over 1000 citations), and form basis for extracting scientific knowledge from measurement data. This collection is augmented by well-established databases such as PDB, PubChem, ChEMBL and others.
To perform reproducible computations, our group develops and maintains software tools that are capable of utilizing the Crystallographic Information Framework. These tools are routinely used to ensure the syntactic and semantic validity of data in the COD as well as other projects. Our group also routinely collaborates with the International Union of Crystallography and has contributed to the development of the CIF2 file format and the DDLm dictionary definition language.
Current project is ”Chemical annotation in the Crystallography Open Database (COD)”, 2020–2022 (S-MIP-20-21, project leader - dr. A. Merkys).
1. Vaitkus, A., Merkys, A. & Gražulis, S. Validation of the Crystallography Open Database using the CIF framework. Journal of Applied Crystallography. 2021, accepted.
2. Gražulis, S., Merkys, A., Vaitkus, A., Chateigner, D., Lutterotti, L., Moeck, P., et al., Le Bail, A. Crystallography open database: history, development, and perspectives. In: O. Isayev, A. Tropsha, & S. Curtarolo (Eds.), Materials Informatics. 2019, 1–39. Wiley. doi:10.1002/9783527802265.ch1 .
3. Mendili, Y. E., Vaitkus, A., Merkys, A., Gražulis, S., Chateigner, D., Mathevet, F., et al. Guen, M. L. Raman Open Database: first interconnected Raman–X-ray diffraction open-access resource for material identification. Journal of Applied Crystallography. 2019, 52(3): 618–625. doi:10.1107/s1600576719004229 .
4. Quirós, M., Gražulis, S., Girdzijauskaitė, S., Merkys, A., & Vaitkus, A. Using smiles strings for the description of chemical connectivity in the crystallography open database. Journal of Cheminformatics. 2018, 10(23). doi:10.1186/s13321-018-0279-6.
5. Merkys, A., Mounet, N., Cepellotti, A., Marzari, N., Gražulis, S., Pizzi, G. A posteriori metadata from automated provenance tracking: integration of AiiDA and TCOD. Journal of Cheminformatics. 2017, 9(1): 56. doi:10.1186/s13321-017-0242-y.
Crystallographic Data Validation
The crystallographic data validation topic concerns collection, analysis and validation of crystallographic information in the COD. As experimental crystallographic data is not directly usable in computational chemistry analyses, additional information and assumptions have to be employed to augment the crystallographic data with chemical annotations in fully automated manner. Analysis of the results derived by such processes leads to the identification of outliers, which may be genuine either due to the problems with computation workflows or the data itself. Identification of the latter is crucial to increase the quality of both the crystallographic and the chemical data in the COD as well as other bodies of experimental crystallographic data (project leader - A. Merkys).
Fig. 1. Distribution of c(cCH)2(H)-c(cCH)2(H)-c(cCH)2(H) bond angles in the COD data.
Derivation of Chemical Information from Crystallographic Data
The emergence of new interdisciplinary fields has stipulated the need to establish a greater connectivity between scientific data from different research areas. One strategy of relating crystallographic data to other fields such as chemistry or material science relies on generating chemical descriptors of molecules from their crystallographic structures and using these descriptors to identify the chemical compounds that the crystals encompass. To facilitate the cross-linking of the COD with other open resources, our group has developed an automated pipeline capable of extrapolating chemical data such as atom connectivity, bond orders and atom charges from crystallographic models, thus enabling the generation of chemical descriptors. These descriptors were later used to link a large portion of the COD to the PubChem database (project leader - A. Vaitkus).
Fig. 2. Schema of the automated pipeline used to derive chemical information from the COD.
Molecular Geometries in Macromolecular Structures
Identifying the probable positions of the protein side-chains is one of the protein modelling steps that can improve the prediction of protein-ligand, protein-protein interactions. In our research, we are trying to approach rotamer library generation problem by scanning for side-chain conformations and calculating potential energy values instead of pooling occurrences of angles only from the structural data (PDB). This enables to study side-chains regardless of unobserved angles or modified amino acids. The flexibility of the method enhances the study of possible side-chain positions and their potential interactions with the ligands (project leader - A. Grybauskas).
Fig.3. Rotamer generation steps: first, the energy values of all angles are calculated until they reach certain threshold (dead-end elimination), then the lowest energy rotamers are kept.
Mechanisms of Flavoenzyme Redox Reactions
Flavoenzymes contain flavinmononucleotide (FMN) or flavinadenindinucleotide (FAD) in their active centres. The distinctive feature of flavoenzymes is their ability to transform single-electron transfer into a two-electron one. They play important roles in biological oxidation-reduction, hydroxylation, transhydrogenation, antioxidant protection, redox signalling, and other processes. Flavoenzymes also participate in biodegradation of toxic environmental pollutants and manifestation or neutralization of therapeutic activity/cytotoxicity of drugs or xenobiotics. Frequently, flavoenzymes are considered as drug targets. Taken together, these factors foster the permanent interest in the studies of flavoenzyme catalysis and its application in biomedicine, industries, and environmental protection. During last two decades, our studies were concentrated on the following issues: 1) the mechanisms of electron/hydride transfer in catalysis of flavoenzymes electrontransferases and transhydrogenases; 2) single- and two-electron reduction of quinones, nitroaromatics and other redox active organic compounds by mammalian, microbial or parasite flavoenzymes and their impact on their cytotoxicity. These studies were accompanied by intensive synthesis of above compounds; and 3) studies of prooxidant xenobiotics as inhibitors and subversive substrates for antioxidant mammalian or parasite FAD/SS and FAD/SS/SeS-containing enzymes.
Our main activities in 2017–2020 were as follows: a) characterization of interaction mechanism of quinones, nitroaromatics and aromatic N-oxides with possible target enzymes in bacteria (S. aureus flavohemoglobin, collaboration with Dr L. Baciou and F. Lederer, Université Paris-Sud, France), mammalian cells (neuronal NO synthase, collaboration with Dr J.-L. Boucher, Université Paris Descartes, France), and parasites (Plasmodium falciparum ferredoxin:NADP+ oxidoreductase, collaboration with Dr A. Aliverti, Universitá degli Studi di Milano, Italy); b) characterization of the mechanisms of two-electron reduction of quinones and nitroaromatics compounds by E. coli nitroreductase A and other nitroreductases (collaboration with Dr D. F. Ackerley, Victoria University of Wellington, New Zealand); c) evaluation of nitroaromatic compounds as inhibitors for Plasmodium falciparum glutathione reductase and Trypanosoma congolense trypanothione reductase in the context of development of antiplasmodial and antitrypanosomal agents (collaboration with Dr E. Davioud-Charvet, Université de Strasbourg, France, and Dr J. S. Blanchard, Albert Einstein College of Medicine, NY, USA); d) continuation of synthesis, studies of enzymatic single- and two-electron reduction and mammalian cell culture cytotoxicity studies of new polynitrobenzenes, nitrofurans, nitrothiophenes, and aromatic N-oxides (EU Structural Funds, Global Grant Measure, Grant No. 09.3.3-LMT-K-712-01-0058, 2018–2021). In 2020, we are continuing the studies of a potent antiplasmodial agent, 1,4-naphthoquinone plasmodione, and its derivatives (in collaboration with Dr E. Davioud-Charvet, Lithuanian-French Programme “Gilibert” , No. S-LZ-19-4). In addition, we have carried out the studies for antibacterial activity of aromatic di-N-oxide compounds used as single agents and in combination with conventional antibiotics, and participated in the studies of antibacterial photodynamic therapy.
1. Nemeikaitė-Čėnienė, A., Šarlauskas, J., Misevičienė, L., Marozienė, A., Jonušienė, V., Lesanavičius, M., Čėnas, N. Aerobic cytotoxicity of aromatic N-oxides: the role of NAD(P)H:quinone oxidoreductase (NQO1). Int. J. Molec. Sci. 2020, 21: 8754.
2. Lesanavičius, M., Aliverti, A., Šarlauskas, J., Čėnas, N. Reactions of Plasmodium falciparum ferredoxin:NADP+ oxidoreductase with redox cycling xenobiotics: a mechanistic study. Int. J. Molec. Sci. 2020, 21: 3234.
3. Nemeikaitė-Čėnienė, A., Šarlauskas, J., Jonušienė, V., Marozienė, A., Misevičienė, L., Yantsevich, A. V., Čėnas, N. Kinetics of flavoenzyme-catalyzed reduction of tirapazamine derivatives: implications for their prooxidant cytotoxicity. Int. J. Molec. Sci. 2019, 20: 4602.
4. Marozienė, A., Lesanavičius, M., Davioud-Charvet, E., Aliverti, A., Grellier, P., Šarlauskas, J., Čėnas, N. Antiplasmodial activity of nitroaromatic compounds: correlation with their reduction potential and inhibitory action on Plasmodium falciparum glutathione reductase. Molecules. 2019, 24: 4509.
5. Moussaoui, M., Misevičienė, L., Anusevičius, Ž., Marozienė, A., Lederer, F., Baciou, L., Čėnas, N. Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin. Free Radic. Biol. Med. 2018, 123: 107–115.
Mechanisms of Antiplasmodial and Antitrypanosomal in vitro Activity of Nitroaromatic Compounds
We found that the in vitro antiplasmodial activity of a series of nitrobenzenes and nitrofurans increases with their single-electron reduction potential (E17), log D, and their ability to inhibit Plasmodium falciparum, but not human erythrocyte glutathione reductase. Ferredoxin:NADP+ oxidoreductase is expected to be the most active enzymatic reductant of nitroaromatics in parasite (Marozienė et al. Molecules. 2019, 24: 4509). Similarly, the in vitro activity of a series of 5-vinylquinoline-substituted nitrofurans against Trypanosoma b. brucei exhibited parallelism with their inhibitory activity against trypanothione reductase (Benitez et al. Chemija. 2020, 31: 111–117).
Structure of erythrocyte glutathione reductase with indicated domains of binding of substrates and inhibitors (Sarma et al. J. Mol. Biol. 2003, 328: 893–907)
Enzymatic Redox Reactions and Cytotoxicity of Aromatic N-oxides
3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) is an anticancer agent selective for hypoxic cells. However, some of its derivatives may exert significant toxicity to normal (oxic) cells. We found that the aerobic cytotoxicity of a number of TPZ derivatives is well above that of quinones with similar E17 values, although their reactivity towards single-electron transferring flavoenzymes is similar. This was attributed to the potentiation of cytotoxicity of TPZ derivatives by cytochromes P-450 and NAD(P)H: quinone oxidoreductase (NQO1). The contribution of NQO1, which reduces TPZ derivatives in a mixed single- and two-electron way, is statistically significant (Nemeikaitė-Čėnienė et al. Int. J. Molec. Sci. 2019, 20: 4602; Int. J. Molec. Sci. 2020, 21: 8574).
Scheme of single-electron reduction, redox cycling, and formation of metabolites of tirapazamine (1)
Antibacterial Activity of Aromatic di-N-oxides
Tirapazamine (TPZ) and its analogues were active with respect to both Gram-positive and -negative bacterial strains. The combination of TPZs with ciprofloxacin and nitrofurantoin (NFT) could generate additive and synergistic effects. These findings suggest that aromatic di-N-oxides may become valuable antibiotic complements (Polmickaitė-Smirnova et al. Appl. Sci. 2020, 10(12): 4062).
Isobolograms of combinatory action of TPZ and NFT (Polmickaitė-Smirnova et al., 2020).
Microbial Biochemical Diversity as a Source of New Biocatalysts
Microbial Biochemical Diversity as a Source of New Biocatalysts
Modern biotechnology is based on the application of enzymes derived predominantly from microorganisms. Both genetic and biochemical microbial diversity is an immense source of different proteins and biocatalysts. The analysis and exploration of said diversity is one of the main aims of our group. The studies are concentrated on several fields. The first one is related to the isolation of N-heterocyclic compound-utilizing microorganisms and the investigation of the catabolic pathways of these compounds in individual bacteria. Unique oxygenases active towards indole, pyridine and 4-hydroxypyridine as a primary substrate have been characterized, genetically modified and applied for development of biocatalytic processes [1–4]. Screening for novel enzymes is also carried out by applying metagenomic techniques – effective selection systems combined with tailored substrates .
More than 160 of differently modified nucleotides play a crucial role in various biological processes. Also, various modified nucleotides are used as promising building blocks for programmable changes of nucleic acids. The biosynthetic pathways of many modified nucleotides including N4-acetylcytosine derivatives are well understood, but the catabolism or salvage of those compounds are only scarcely studied. For the first time, we show that in E. coli the ASCH domain-containing protein YqfB, which has a unique Thr-Lys-Glu catalytic triad, catalyses the hydrolysis of N4-acetylcytidine. In addition, novel N4 amino acid-acylated and alkylated 2'-deoxycytidine analogues were synthesized.
1. Časaitė et al. Microbial degradation of pyridine: a complete pathway deciphered in Arthrobacter sp. 68b. Appl. Environ. Microbiol. 2020, 86: e00902-20.
2. Sadauskas et al. Bioconversion of biologically active indole derivatives with indole-3-acetic acid-degrading enzymes from Caballeronia glathei DSM50014. Biomolecules. 2020, 10: 663.
3. Vaitekūnas et al. Biochemical and genetic analysis of 4-hydroxypyridine catabolism in Arthrobacter sp. strain IN13. Microorganisms. 2020, 8: 888.
4. Sadauskas et al. Enzymatic synthesis of novel water-soluble indigoid compounds. Dyes Pigm. 2020, 173: 107882.
5. Urbelienė et al. A rapid method for the selection of amidohydrolases from metagenomic libraries by applying synthetic nucleosides and a uridine auxotrophic host. Catalysts. 2020, 10: 445.
Microbial Degradation of Pyridine: A Complete Pathway Deciphered in Arthrobacter sp. 68b
Pyridine and its derivatives constitute majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to the environmental pollution. It is known that they can be degraded by various bacteria in the environment, however, the degradation of unsubstituted pyridine has not yet been completely resolved. Here, we present data on the pyridine catabolic pathway in Arthrobacter sp. 68b at the level of genes, enzymes and metabolites. The pyr genes cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalysed by a two-component flavin-dependent monooxygenase system, encoded by pyrA and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation (Časaitė et al. Appl. Environ. Microbiol. 2020, 86: e00902-20).
In vitro reconstruction of the pyridine degradation pathway
(a) The analysis of pyridine metabolites by LC-MS and the metabolic pathway of pyridine. Samples were analysed in the positive or negative ionization mode; the extracted ion chromatograms correspond to the quasimolecular ions of (1) pyridine (m/z=80 [M+H]+), metabolite 2 – (Z)-N-(4-oxobut-1-enyl)formamide (m/z=114 [M+H]+), metabolite 3 – (Z)-4-formamidobut-3-enoic acid (m/z=130 [M+H]+), 4 – succinic acid semialdehyde (m/z=101 [M-H]–), and 5 – succinate (m/z=117 [M-H]–);
(b) purified proteins participating in pyridine pathway;
(c) pyr gene cluster in the p2MP plasmid. PyrA – pyridine monooxygenase, PyrE and FR – flavin reductase, PyrB – (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, PyrC – amidohydrolase, PyrD – succinate semialdehyde dehydrogenase.
YqfB Protein from Escherichia coli: Atypical Amidohydrolase Active towards N4-acylcytosine Derivatives
Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid hypothetical protein YqfB from E. coli is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N4-acetylcytidine (ac4C). YqfB has several interesting and unique features: (i) it is the smallest monomeric amidohydrolase, (ii) it is active towards structurally different N4-acylated cytosines/cytidines, iii) it has a very high activity rate (kcat/Km up to 2.8×106 M–1s–1) and (iv) it contains a unique Thr-Lys-Glu catalytic triad and Arg acting as an oxyanion hole. YqfB ability to hydrolyse various N4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases. (Stanislauskienė et al. Sci. Rep. 2020, 10: 788).
Predicted structure of the active centre of YqfB and proposed catalytic mechanism of ac4C hydrolysis
(a) The overall structure of the proposed enzyme-substrate complex.
(b) A detailed view of Yqfb active centre with bound ac4C, as generated by MD simulation; dashed lines correspond to hydrogen bonds.
(c) The acylation step of ac4C hydrolysis catalysed by YqfB.
Bioconversion of Biologically Active Indole Derivatives with Indole-3-Acetic Acid-Degrading Enzymes from Caballeronia glathei DSM50014
A plant auxin hormone indole-3-acetic acid (IAA) can be assimilated by bacteria as an energy and carbon source, although no degradation has been reported for indole-3-propionic acid and indole-3-butyric acid. Caballeronia glathei possesses a full iac gene cluster and is able to use IAA as a sole source of carbon and energy. Next, IacE is responsible for the conversion of 2-oxoindole-3-acetic acid intermediate into the central intermediate 3-hydroxy-2-oxindole-3-acetic acid (DOAA). Finally, IacA and IacE were shown to convert a wide range of indole derivatives, including indole-3-propionic acid and indole-3-butyric acid, into corresponding DOAA homologs. This work provides novel insights into Iac-mediated IAA degradation and demonstrates the versatility and substrate scope of IacA and IacE enzymes. (Sadauskas et al. Biomolecules. 2020, 10: 663).
Comparison of biodegradation of indole in Acinetobacter sp. strain O153 and biodegradation of IAA in Caballeronia glathei DSM50014 (M. Sadauskas, PhD thesis).
Grey reaction arrows indicate spontaneous reactions. Arrows with identical colours indicate genes and proteins with similar predicted functions.
Protein Structural Bioinformatics
Protein Structural Bioinformatics
Proteins typically function as three-dimensional (3D) structures, often through interaction with each other and/or with other macromolecules. Protein 3D structure is also the most conserved property of evolutionary related proteins. Therefore, the knowledge of structures of individual proteins and their complexes is essential for understanding their evolution, function and molecular mechanisms. However, the experimental determination of protein structure is slow, expensive and not always successful. The increasing computer power and the flood of biological data make computational prediction of 3D structure of proteins and their complexes an important alternative to experiments. Computational methods are also indispensable in the analysis or prediction of interaction sites even in the case of experimentally solved structures. However, computational methods have their own challenges. Computational structure prediction works best when related structures (templates) are available. Therefore, the detection of remote homology is one of the major impediments. The reliable estimation of the accuracy of predicted structures is another important problem. More efficient methods for the analysis and prediction of protein binding sites are also badly needed.
Our team addresses a broad range of protein-centred research topics that can be collectively described as Computational Studies of Protein Structure, Function and Evolution. There are two main research directions:
1) Development of computational methods for detection of protein homology, for comparative protein structure modelling, and for analysis and evaluation of 3D structure of proteins and protein complexes. In recent years, we have developed several new methods addressing these research topics. All of the software packages implementing these methods are freely available at our web site (http://bioinformatics.lt/software).
2) Application of computational methods to biological problems. In this research direction, we have been using computational methods for discovering general patterns in biological data, structural/functional characterization of proteins and their complexes, design of novel proteins and mutants with desired properties. Over the years, our major focus has been on studies of DNA replication and repair systems in viruses, bacteria and eukaryotes. In addition, we have entered a highly dynamic CRISPR-Cas research field and have already made important contributions in elucidating structural and mechanistic properties of CRISPR-Cas systems and their evolutionary relationships.
1. Makarova et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020, 18(2): 67–83. doi:10.1038/s41579-019-0299-x.
2. Gasiunas et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat Commun. 2020, 11(1): 5512. doi:10.1038/s41467-020-19344-1.
3. Kazlauskas et al. Diversity and evolution of B-family DNA polymerases. Nucleic Acids Res. 2020,; 48(18): 10142–10156. doi:10.1093/nar/gkaa760.
4. Dapkūnas, J., Olechnovič, K. & Venclovas, Č. Structural modeling of protein complexes: Current capabilities and challenges. Proteins. 2019, 87(12): 1222–1232. doi:10.1002/prot.25774.
5. Olechnovič, K. & Venclovas, Č. VoroMQA web server for assessing three-dimensional structures of proteins and protein complexes. Nucleic Acids Res. 2019, 47(W1): W437–W442. doi:10.1093/nar/gkz367.
Predicting Structures of Protein Assemblies in Global CASP and CAPRI Experiments
Measuring progress in ability to predict 3D structures of protein complexes is the goal of CAPRI experiments and, lately, one of the major aims of CASP experiments. In summer of 2020, we participated in both CASP14 and CAPRI experiments that were executed in parallel. In both experiments, we tested the performance of our template-based modelling, free docking and hybrid modelling protocols. Among key components of these protocols were the latest versions of PPI3D and VoroMQA methods, developed in our group. The PPI3D web server enables searching, analysing and modelling protein complexes, whereas VoroMQA allows estimation of protein structure quality. Independent assessors found our CAPRI results (group ‘Venclovas’) to be at the top jointly with the results of the other two groups. According to CASP assessment, our group was second. CASP and CAPRI experiments were performed on different test sets, and assessment methods were somewhat different. Despite slight differences in overall ranking, our group continued to be one of the leading groups in the modelling of protein complexes. The results of the joint CASP14-CAPRI experiment are to be published in a special issue of Proteins.
Uncovering Convoluted Evolutionary History of Human Replicative Polymerases
B-family DNA polymerases (PolBs) are the most common replicases, present in all domains of life and in many DNA viruses. Despite extensive research, origins and evolution of PolBs remain enigmatic. To unravel the evolutionary history of PolBs, we performed comprehensive computational analysis of these proteins originating from archaea, bacteria, eukaryotes and viruses. As a result, we defined and characterized six new groups of archaeal PolBs and a new group of bacterial PolBs, which appears to be related to the catalytically active N-terminal module of the eukaryotic Polε. We also uncovered the similarity of the catalytically inactive Polε C-terminal module to Polα. Finally, we discovered that two novel groups of archaeal PolBs have C-terminal metal-binding domains, closely related to those present in eukaryotic Polα and Polε. Collectively, the results of this study allowed us to propose a scenario for the evolution of human and other eukaryotic PolBs.
Phylogenetic tree of B-family DNA polymerases
(Kazlauskas et al. Nucleic Acids Res. 2020, 48: 10142–10156).
Surveying CARF and SAVED Proteins, Key Players in Antivirus Defence of Prokaryotes
Proteins possessing CARF and SAVED domains are key components of cyclic oligonucleotide-based antiphage signalling systems (CBASS) that, upon activation, induce cell dormancy or death. Most CARF proteins belong to a CBASS built into type III CRISPR–Cas systems. The CARF domain binds cyclic oligoA (cOA) synthesized by the Cas10 polymerase-cyclase and allosterically activates the effector, typically a promiscuous ribonuclease. Some CARF domains also function as ring nucleases that cleave cOA thereby terminating signal transduction. Due to the extreme sequence divergence and the diversity of domain architectures of CARF domain-containing proteins, CARF domains are often overlooked or misannotated in genome analyses. Therefore, we performed a comprehensive analysis of the CARF and SAVED domains encoded in bacterial and archaeal genomes. Based on this analysis, we proposed a classification of CARF and SAVED proteins, predicted several families of novel ring nucleases, and provided new insights into the organization of the cOA signalling pathway (Makarova et al. Nucleic Acids Res. 2020, 48: 8828–8847).
CARF domain structures (A) Topology of the CARF fold (B) Superposition of multiple CARF domain structures coloured by chain progression.
Protein Structure and Interactions in Phospholipid Membranes
Protein Structure and Interactions in Phospholipid Membranes
The molecular organisation and function of biological membranes are essential to the understanding of living processes in general and the development of various biotechnological processes including molecular medicine in particular. Membrane proteins (MPs) represent almost 60% of pharmaceutical targets. However, despite their fundamental role, only 2% of the protein of known structure are that of MPs, and such lack of knowledge seriously affects understanding of the membrane protein functions slowing down the development of new diagnostic tools and therapies. The major difficulties and challenges for structural and functional studies of MPs arise from their instability outside a lipid bilayer environment, where specific hydrophobic and other molecular forces keep the protein in its native and active conformational state. Therefore, considerable efforts are directed towards the development of simplified but biologically relevant model membrane systems to study molecular processes in membranes.
Our group is specializing in the development of tethered bilayer membrane (tBLM) models. tBLMs are solid-supported phospholipid bilayers anchored to a surface via hydrophobic interactions between the molecular anchors and hydrophobic sheet of the membrane. The molecular anchors are synthetic thiolipids or silanes covalently attached to metal or metal-oxide surfaces. The anchors may contain hydrophilic fragments separating thiol/silane group and the glycerol backbone of the lipid, thus ensuring 1–2 nm thick water-reservoir between tethered bilayer and solid support. Alternatively, bilayers with no water sub-phase can be engineered. Recently, we developed an affordable and reproducible methodology for tBLM assembly using multilamellar vesicle fusion. We showed that such tBLMs are capable of reconstituting transmembrane proteins retaining their biological function. Membrane reconstituted proteins (peptides, oligomers) may be probed by the surface specific techniques, including surface plasmon resonance, vibrational spectroscopies and atomic force microscopy. Fine structural details revealing the molecular geometry of tBLMs are evaluated by the neutron reflectometry. Functional properties membranes with reconstituted protein complexes are accessible by the electrochemical impedance spectroscopy (EIS). The theoretical framework of EIS developed in our group allows a detailed analysis of protein membrane interactions as well as applications of tBLMs for bioanalysis.
- Tumenas, S., Ragaliauskas, T., Penkauskas, T., Valanciute, A., Andriulevicius, F., Valincius, G. Solvent effects on composition and structure of thiolipid molecular anchors for tethering phospholipid bilayers. Applied Surface Science. 2020, 509: 145268.
- Talaikis, M., Valincius, G., Niaura, G. Potential-induced structural alterations in the tethered bilayer lipid membrane-anchoring monolayers revealed by electrochemical surface-enhanced Raman spectroscopy. J.Phys.Chem. C. 2020, 124(35): 19033–19045.
- Penkauskas, T., Zentelyte, A., Ganpule, S., Valincius, G., Preta, G. Pleiotropic effects of statins via interaction with the lipid bilayer: A combined approach. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2020, 1862(9): 183306.
- Raila, T., Ambrulevicius, F., Penkauskas, T., Jankunec, M., Meškauskas, T., Vanderah, D .J., Valincius, G. Clusters of protein pores in phospholipid bilayer membranes can be identified and characterized by electrochemical impedance spectroscopy. Electrochimica Acta. 2020, 364: 137179.
Properties and Function of Tethered Bilayer Membranes (tBLM), a biomimetic model of phospholipid membrane, depends on the structure of the self-assembled lipid-like monolayer that anchors tBLM to the surface. Electrode potential that can be adjusted via an external source is a physical variable, which can be used to fine-tune structure and possibly the function of tBLMs. In this project, we explored the potential-induced structural changes in monolayers composed of widely-used in tBLM design lipid-like long-chain WC14 molecules and short-chain hydrophilic backfiller 2 mercaptoethanol by the electrochemical surface-enhanced Raman spectroscopy. To infer the electric potential-induced changes in monolayer, the metal−adsorbate (i.e. Au–S and Au–O) spectral bands and C−C stretching vibrational mode of WC14's alkyl chains in all-trans configuration near 1130 cm-1 were analysed. Negative electric potential promotes the mobility of anchor molecules on the surface by decreasing the strength of metal−adsorbate bonding. At the same time, water pushes the hydrophobic WC14’s chains into phase-segregated clusters and in such a way minimizes the system’s energy. The clustering of molecular anchors has a detrimental effect on the integrity of tBLMs and their electrical insulation, therefore we propose the spectral band near 1130 cm-1 as a predictor of the functional properties of tBLMs. In general, our findings explain detrimental effects of negative going electric polarization of tBLMs empirically observed by a number of researchers before. This work was published in Talaikis et al. J. Phys. Chem. C. 2020, 124: 19033–19045).
Pleiotropic Effects of Statins via Interaction with the Lipid Bilayers
Statins are selective inhibitors of cholesterol biosynthesis, used worldwide for cholesterol lowering in the primary and secondary prevention of cardiovascular diseases. Clinical trial studies indicated that the observed general benefits of statins appear to be greater than what might be expected from changes just in lipid levels, suggesting effects beyond cholesterol lowering (i.e. pleiotropic effects). Using a combined approach based on biophysical and biological methods, we demonstrate that lipophilic, but not hydrophilic statins are capable of reducing the damage caused by pneumolysin, a toxin of the cholesterol-dependent cytolysins (CDCs) family. This protection correlates with statins’ lipophilicity (expressed by the log P value) and capacity to interact with the lipid bilayer. Our data suggest that lipophilic statins associate with membranes and interfere with the ability of CDCs to bind to membrane cholesterol, influencing membrane lipid rafts organization. The ability to influence membrane lipid structure is one of the reported cholesterol-independent effects of statins. Evaluation of the capacity of statins to modulate membrane properties is an essential step for developing a correct therapeutic approach for cardiovascular diseases as well as for understanding the potential of this class of drugs in cancer therapy to increase tumour response to cytotoxic agents. The work was published in Penkauskas et al. BBA-Biomembranes. 2020, 1862(9): 183306.
Cluster formation is a widely-observed phenomenon, which results in unique sets of properties both in physical, chemical, and biological domains of nature. We found a mathematical description of clustering of membrane defects, specifically, of ion-conducting protein pores in tethered phospholipid bilayers. By invoking the Voronoi tessellation concept we demonstrate the possibility to distinguish between random and sparsely clustered patterns both in computer-generated and real-world systems using one single parameter σ, the standard deviation of the normalized Voronoi sector areas distribution. For random systems, σ0.54, and for clustered patterns σ>0.54. Because of a specific structure and dielectric properties of tethered bilayers, they can be characterized by an alternating current technique, electrochemical impedance spectroscopy (EIS). EIS measures macroscopic parameters of systems, and it is not a structural technique per se. However, we found the EIS spectra-derived quantitative metric ζ to be a diagnostic parameter that allows assessment of the distribution type (homogeneous, random or clustered) of defects at nanometre level. One of the most interesting findings of the current study is the fact that the EIS derived ζ parameter is sensitive to an average size of the defects, thus, enabling a purely electrochemical methodology to access fine structural information such as the size of incomplete protein pores in phospholipid bilayers. Overall, our results demonstrate a fundamental property of the macroscopic technique, electrochemical impedance spectroscopy, to probe structural arrangement of defects with sizes between 0.5 nm to 25.5 nm located in a thin, 2 nm thick phospholipid dielectric layer. This project is a part of collaboration efforts with Dr. Tadas Meškauskas’ group from the Faculty of Mathematics and Informatics at Vilnius University. It was published in Raila et al. Electrochimica Acta, 2020, 364: 137–179.
Signalling in Prokaryotic Antiviral Defence
Fig.1. Type III CRISPR-Cas immunity mechanism.
Signalling in Prokaryotic Antiviral Defence
Cyclic mono- and di-ribonucleotides are widely employed for controlling various biological processes by eukaryotes and especially prokaryotes. Purine ribonucleotides serve not only as building blocks for RNA, universal currency of energy and components of coenzymes such as NAD(P)+, FAD or CoA, but are also assembled into signalling molecules. Both prokaryotes and eukaryotes employ cyclic AMP (cAMP) and cGMP as key second messengers in a variety of biological processes, including quorum sensing, energy homeostasis, neuronal signalling, and muscle relaxation. Prokaryotes also use a variety of cyclic dinucleotides – c-di-AMP, c-di-GMP, and 3’3’-cGAMP – as second messengers for biofilm formation, virulence and regulation of bacterial cell cycle. Recent studies in bacteria have reported that various cyclic oligonucleotides are used as signalling molecules in bacterial antiviral defence systems of Type III CRISPR-Cas and CBASS (cyclic-oligonucleotide-based anti-phage signalling systems). As these systems are widespread and abundant, this suggests that signalling is widely used in prokaryotic antiviral defence.
Our group has pioneered by elucidating the interference mechanism of the Type III CRISPR-Cas immunity 1–4. We revealed that to provide immunity against invading nucleic acids in prokaryotes, Type III CRISPR-Cas system combines transcription-dependent DNA degradation by crRNA guided Csm or Cmr complex [1–3] with the cyclic oligoadenylates (cAn)-dependent immunity pathway [3–5]. In response to the viral RNA binding the Csm/Cmr complex synthesizes cAn molecules of various ring size (n=2–6) [4,5]. The cA4 or cA6 acts as a signalling molecule that binds to the CARF domain sensor of the stand-alone Csm6 or Csx1 proteins and allosterically activates the non-specific ribonucleolytic activity of their HEPN effector domains [4,5]. Bioinformatics analysis revealed that the sensor CARF and SAVED (a divergent version of CARF) domains are found fused with diﬀerent enzymatic or non-enzymatic effector domains of Type III CRISPR-Cas associated or CRISPR-Cas unrelated proteins. We aim to understand the molecular and structural mechanisms by which CARF/SAVED domain-containing proteins are involved in bacterial immunity or possibly other cell processes.
- Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, Č., Siksnys, V. Spatiotemporal control of type III-A CRISPR-Cas immunity: coupling DNA degradation with the target RNA recognition. Molecular Cell. 2016, 62: 295–306.
- Tamulaitis, G., Venclovas, Č, Siksnys V. Type III CRISPR-Cas immunity: major differences brushed aside. Trends in Microbiology. 2017, 25(1): 49–61.
- Mogila, I., Kazlauskiene, M., Valinskyte, S., Tamulaitiene, G., Tamulaitis, G., Siksnys, V. Genetic dissection of the type III-A CRISPR-Cas system Csm complex revealsroles of individual subunits. Cell Reports. 2019, 26(10): 2753–2765, e4.
- Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G., Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science. 2017, 357: 605–609.
- Smalakyte, D., Kazlauskiene, M., Havelund, J.F., Rukšėnaitė, A., Rimaite, A., Tamulaitiene, G., Færgeman, N. J., Tamulaitis, G., Siksnys, V. Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic Acids Research. 2020, 48(16): 9204–9217.
Single-molecule studies of protein and DNA interactions
Single-molecule studies of protein and DNA interactions
Real-time monitoring of single-proteins on nucleic acid (NA) substrates is an essential tool for studying NA-interacting proteins allowing better mechanistic understanding. To follow individual proteins on a NA molecule, one or both ends of NA molecule that are extended by an external force are attached to the surface. Over the past 20 years, a number of such methods emerged: tethered particle motion (TPM), optical or magnetic tweezers (OTs or MTs), flow-stretch assays and various combinations of these methods. Stretched NAs is a common means to investigate the dynamics of protein-NA interactions. Several experimental strategies are employed to extend and align NA molecules (e.g. NA combing or through hydrodynamic flow). The hydrodynamic flow method allows attaching NA molecules to the glass surface, where they are stretched by a shear hydrodynamic flow. The flow can be applied to extend single-end tethered NA, or deployed to allow the specific double-end tethering. Anchoring the NA onto a lipid bilayer and a diffusion barrier etched on the microscope slide causes the alignment of the NA moving under a buffer flow. This method, known as DNA curtains, allows imaging of many DNA molecules in parallel.
Recently our team developed an alternative assay to the original DNA Curtains that we termed the “Soft” DNA Curtains (LRC grant No. S-MIP-17-59). We fabricated streptavidin patterns (i.e. line-features) on the modified coverslip surface that can be utilized to assemble stably immobilized biotinylated DNA arrays. The application of hydrodynamic buffer flow allows extension of the immobilized DNA molecules along the surface of the flow cell channel. We also fabricated the uniformly oriented double-tethered DNA Curtains using heterologous labelling of the DNA by biotin and digoxigenin. We increased the stability of the immobilized DNA molecules using a more stable alternative to sAv called traptavidin as an ink for the fabrication of protein templates.
The main goal of our research topic is to apply the developed platform for studies of DNA targeting mechanisms of diverse CRISPR-Cas systems family (LRC grant No. S-MIP-20-55), novel molecular-tools – prokaryotic Argonaute (pAgo) proteins (LRC grant No. 09.3.3-LMT-K-712-19-0113) and various restriction endonucleases. Our newest publication on the “Soft” DNA Curtains  and its continuation  received a broad interest of scientists from various fields.
- Tutkus, M., Rakickas, T., Kopūstas, A., Ivanovaitė, Š., Venckus, O., Navikas, V., Zaremba, M., Manakova, E., Valiokas, R. Fixed DNA molecule arrays for high-throughput single DNA-protein interaction studies. Langmuir. 2019, 35, 17: 5921–5930.
- Kopūstas, A., Ivanovaitė, Š., Rakickas, T., Pocevičiūtė, E., Paksaitė, J., Karvelis, T., Zaremba, M., Manakova, E., Tutkus, M. bioRxiv. doi.org/10.1101/2020.06.15.151662 (in press).
- Tutkus, M., Marciulionis, T., Sasnauskas, G., Rutkauskas, D. DNA-endonuclease complex dynamics by simultaneous FRET and fluorophore intensity in evanescent field. Biophysical Journal. 2017, 112(5): 850–858.
- Tutkus, M., Sasnauskas, G., Rutkauskas, D. Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET. Biopolymers. 2017, 107(12): e23075.
Protein X-ray crystallography is the primary technique for elucidation of three-dimensional protein structures, which are critical for understanding macromolecule mechanism and function.
We perform protein crystallization using Oryx8 and Gryphon crystallization robots, monitor crystal growth in automatic Rigaku Minstrel DT UV stations and collect X-ray diffraction data either on an in-house Rigaku MicroMax-007HF X-ray diffractometer fitted with a Dectris Pilatus 3R 200K-A detector or during data collection sessions in DESY synchrotron, Hamburg. A 200 kV Cryo-EM Glacios microscope is currently being installed at the Life Sciences Center.
Our research combines two major directions:
1) Structural characterization of prokaryotic proteins and protein complexes involved in bacterial antiviral defence. In order to survive under a constant pressure of phage infection, bacteria have developed a great variety of defence mechanisms. Currently, we study components of various bacterial antiviral systems, including:
(i) restriction endonucleases (REases), components of Restriction-Modification systems that protect host bacteria by cleaving bacteriophage DNA, constitute a large and highly diverse family of proteins, which differ in their activity regulation, DNA recognition and DNA cleavage mechanisms. We study orthodox Type II enzymes (PfoI , Kpn2I, AgeI, BsaWI), ATP-dependent REases (NgoAVII, CglI), and REases specific for methylated DNA sequences (LpnPI, EcoKMcrBC , EcoKMcrA , the latter in collaboration with Prof. M. Bochtler group from the International Institute of Molecular and Cell Biology in Warsaw);
(ii) components of the CRISPR-Cas adaptive immunity systems (Cas1-Cas2, Cas6, Cascade);
(iii) bacterial toxin-antitoxin systems, e. g. the MNT-HEPN system from A. flos-aquae cyanobacteria ;
(iv) prokaryotic argonaute proteins and other novel antiviral systems.
2) Crystallographic studies of protein-inhibitor complexes, which are primarily focused on inhibitors of human carbonic anhydrases (hCAs) developed in the Department of Biothermodynamics and Drug Design. hCAs are present in 12 active isoforms in all human tissues. Some isoforms are important therapeutic targets. Design of isoform-specific inhibitors of hCA is a complex project that combines comprehensive thermodynamic description of the protein-ligand interaction with structural characterization. We perform structural characterization of hCA complexes with newly designed inhibitors in order to correlate the binding modes with the thermodynamic parameters of interaction.
1. Tamulaitiene, G., Manakova, E., Jovaisaite, V., Tamulaitis, G., Grazulis, S., Bochtler, M., Siksnys, V. Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family. Nucleic Acids Research. 2019, 47: 997–1010.
2. Zagorskaitė, E., Manakova, E., Sasnauskas, G. Recognition of modified cytosine variants by the DNA binding domain of methyl-directed endonuclease McrBC. FEBS Letters. 2018, 592: 3335–3345.
3. Slyvka, A., Zagorskaitė, E., Czapinska, H., Sasnauskas, G., Bochtler, M. Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping. Nucleic Acids Research. 2019, 47: 11943–11955.
4. Songailiene, I., Juozapaitis, J., Tamulaitiene, G., Ruksenaite, A., Šulčius, S., Sasnauskas, G., Venclovas, Č., Siksnys, V. HEPN-MNT toxin-antitoxin system: the HEPN ribonuclease is neutralized by oligoAMPylation. Molecular Cell. 2020, 80: 929–1140.
5. Kazokaitė, J., Kairys., V, Smirnovienė, J., Smirnov, A., Manakova, E., Tolvanen, M., Parkkila, S., Matulis, D. Engineered carbonic anhydrase VI-mimic enzyme switched the structure and affinities of inhibitors. Scientific Reports. 2019, 9: 12710.
Structure and Thermodynamics for Drug Design
Structure and Thermodynamics for Drug Design
Rational drug design attempts to discover a ligand that would bind a disease target protein with high affinity and selectivity over unintended targets to avoid toxicity. Despite significant efforts, the underlying physical forces that determine the protein-ligand recognition are still rather poorly understood.
To help design compounds and predict their affinity to target proteins, we are assembling datasets, where chemical compounds binding to proteins would be characterized, including
(a) the X-ray crystallographic structures of protein-ligand complexes,
(b) the thermodynamics of interaction including the enthalpy, entropy, Gibbs energy, volume, heat capacity and other thermodynamic parameter changes upon binding,
(c) the kinetics of the same protein-ligand binding, including the on- and off-rates.
We are primarily focused on the human family of twelve catalytically active carbonic anhydrase isoforms as a disease protein-target. These enzymes have essentially the same fold and a highly similar shape of the active site suitable for the testing of isoform selectivity.
The group of scientists come from various backgrounds including molecular biologists, biochemists, organic chemists, biophysicists, physicists, computer modellers, biologists and pharmacists. Organic synthesis scientists design and perform the synthesis of novel compounds, molecular and cellular biologists perform the cloning, expression (both in bacterial and in human cell cultures) and purification of target proteins, biothermodynamicists determine the energetics of binding between the synthesized compounds and the target proteins by isothermal titration calorimetry or thermal shift and search for structure-energetics correlations, crystallographers determine the X-ray crystallographic structures of protein-compound complexes, in silico modellers perform compound docking, and the pharmaceutical scientists perform development studies of the effect of compounds in various biological systems including cancer cell cultures, zebrafish and mice.
- Dudutienė, V., Zubrienė, A., Kairys, V., Smirnov, A., Smirnovienė, J., Leitans, J., Kazaks, A., Tars, K., Manakova, L., Gražulis, S., Matulis, D. Isoform-selective enzyme inhibitors by exploring pocket size according to the lock-and-key principle. Biophys. J. 2020, 119(8): 1513–1524.
- Kazokaitė-Adomaitienė, J., Becker, H. M., Smirnovienė, J., Dubois, L. J., Matulis, D. Experimental approaches to identify selective picomolar inhibitors for carbonic anhydrase IX. Current Medicinal Chemistry. 2020. doi: 10.2174/0929867327666201102112841.
- Zakšauskas, A., Čapkauskaitė, E., Jezepčikas, L., Linkuvienė, V., Paketurytė, V., Smirnov, A., Leitans, J., Kazaks, A., Dvinskis, E., Manakova, E. et al. Halogenated and Di-substituted benzenesulfonamides as selective inhibitors of carbonic anhydrase isoforms. European Journal of Medicinal Chemistry. 2020, 185, 111825.
Compound Binding to Proteins via ‘Induced Fit’ or ‘Lock-and-Key’
Compounds bind to proteins via different mechanisms ranging from ‘induced fit’ to ‘lock-and-key’ modes. The induced fit emphasizes the fact that ligands may alter the protein structure and dynamics upon binding. The ligand-bound and ligand-free states of a protein may differ significantly. The lock-and-key principle is applied when the protein is conformationally rigid and does not visually change its structure upon ligand binding. This seems to us to be the case despite variation in the literature and it is important to design ligands that would fit the active site pocket of the disease target protein as closely as possible.
We have designed a series of compounds with enlarged sizes until they could not fit in the active site and the affinity dropped thousand-fold upon addition of a minor substituent such as a methyl group (see Fig. 1). A series with a substituted hydrophobic group of increasingly larger size was used. If the protein is rigid, there ought to be a limiting size after which the compound cannot fit into the binding site. This size limit should vary for every isoform and could be employed to search for isoform-selective inhibitors. Additionally, the size of the ligand would prevent it from binding to isoforms that are of vital importance and should not be targeted.
The map of correlations between the compound chemical structures and the intrinsic standard Gibbs energy changes upon compound binding to all 12 CA isoforms shows the energies next to the chemical structures (see Fig. 2). Differences in the energies upon binding comparing chemically similar compounds are shown next to the arrows connecting the compounds. Colours represent the 12 catalytically active human CA isoforms.
X-ray crystallographic analysis of several compounds bound to CA II, CA IX and CA XII essentially confirmed that the most successful ligands nearly completely filled the binding site. X-ray crystallography confirmed that the selectivity of 15a towards CA IX was due to the optimal size of the compound and the binding site, e.g. due to the decreased size of residue 131, which has changed from Phe in many CA isoforms to Val in CA IX.
Docking of all compounds to all CA isoforms was employed to test the extent to which we could predict the structural positions and the binding energetic (see Fig. 3). The docking calculations were able to correctly reproduce the binding modes of larger ligands, such as 24, 10, and 15a, with mixed success in reproducing the binding modes of the smaller ligands. For the important CA IX and CA XII isoforms, the docking essentially confirmed conclusions drawn from the X-ray structures. Thus, it has a predictive capability that can be used to design better drugs.