Tomáš Koval

Organophosphorus acid anhydrolase from Alteromonas macleodii: structural study and functional relationship to prolidases.

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacterium Alteromonas macleodii, was prepared recombinantly in Escherichia coli. The crystal structure was determined at 1.8 Å resolution in space group C2, with unit-cell parameters a = 133.8, b = 49.2, c = 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni(2+) ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis.

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

Structure analysis of group I plant nucleases

Structural properties of plant nuclease TBN1 are studied using synchrotron radiation to explain its specificity, role of glycosylation and to contribute to potential application in cancer treatment.

Crystallization of recombinant bifunctional nuclease TBN1 from tomato

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

Crystallization of nepenthesin I using a low-pH crystallization screen

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

Best architecture of protein crystal. Database of protein-polymer interactions showing a unique role of PEG in protein crystallization

Protein molecules regularly ordered in crystal remain highly solvated and are continuously in dynamic equilibrium with solution. The stability of molecules in crystal is ensured by the 3D scaffold formed by intermolecular contacts between adhesive patches of neighbor molecules, whereas 30-80 % of crystal content remains dynamically disordered. That is why the stability of the 3D skeleton, i.e. protein crystal architecture (PCA) is so important. Large surface of protein molecules has usually many adhesive patches and thus we often observe different PCAs of the same protein (polymorphism). Each PCA has its own set of compatible adhesion modes and its own optimal solvent content in crystal. When incompatible adhesion modes are combined during crystal growth, one gets virtually non-diffracting solid phase. Principle of dominating adhesion mode plays a key role in the control of diffraction quality of crystal. It says that well diffracting crystals grow only when incompatible adhesion modes are suppressed. This can be achieved either by rational composition of crystallization solution using protein surface shielding agents (PSSA) [J.Synchr.Radiation (2011)18,50-52] or by chemical modification of the protein surface, or complexation with high affinity ligands. Analysis of already solved structures allows planned strengthening of dominant or weakening of non-compatible adhesion modes and a control of the diffraction quality of growing crystals. Theory of protein-protein adhesion modes shows why the poly(ethyleneglycol type polymers (PEGs) are the most successful precipitants [Z.Kristallogr.(2006)23,613-619]. Database of protein-polymers interactions (DPPI) [Z.Kristallogr.(2011)28,475-480] contains about 4000 of experimentally observed PEG-protein interfaces. It consists of a set of protein structures crystallizing with PEG and the script allowing easy visualization of PEG activities on protein surfaces. Seeing the PEG fragments interfering with protein-protein interfaces, with different types of salts, blocking competitive crystal contacts, protein oligomerization, crystallization, or biological activity, helps to understand the dynamic processes during crystal growth and allows rationalization of its performance on molecular basis. DPPI is available from hasekjh@seznam.cz. The project is supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, CSF project 15-15181S of the CSF, grant LG14009 of MSMT, BioStruct-X (EC FP7 project 283570) and Instruct of ESFRI. Figure 1. PEG induced adhesion mode as a leading motif for crystallization. The insert shows a typical crown ether conformation of polyether caused by interaction of five oxygens to lysine from one side. It induces strong hydrophobic interaction to the neighbor molecule on the opposite side. PDB code 3NBT.

Substrate recognition by non-specific zinc-dependent 3′-nucleases

Jan Dohnalek1, Lars Østergaard2, Petra Lipovova3, Maria Trundova1, Karla Fejfarova1, Jarmila Duskova1, Leona Svecova1, Tereza Skalova1, Jan Stransky1, Tomas Koval1 1Laboratory Of Structure And Function Of Biomolecules, Institute Of Biotechnology, Vestec, Czech Republic, 2Department of Agile Protein Screening, Novozymes A/S, Bagsvaerd, Denmark, 3Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic E-mail: dohnalek@ibt.cas.cz

Changes of LLT1, a ligand for human NKR-P1, with varied glycosylation and crystallization conditions

Natural killer (NK) cells are a type of lymphocytes which kill tumor, virally infected or stressed cells. Decision to kill a cell is made as a result of balance of signals from plenty of activating and inhibitory receptors on surface of the natural killer cells. LLT1 is a ligand expressed primarily on activated lymphocytes (including NK cells itself). It is a binding partner for NKR-P1, receptor on surface of NK cells. Both NKR-P1 and LLT1 have an extracellular part of C-type lectin like (CTL) fold. Receptors and ligands with CTL fold have not been yet excessively studied and their interactions are not still understood. Here we would like to present four crystal structures of LLT1 which we have recently published [1]. LLT1 with homogenous GlcNAc2Man5 glycosylation was expressed in HEK293S GnTIcells [2]. The four LLT1 structures differ by its oligomeric state (monomeric, dimeric and hexameric [three dimers in compact packing]) under various pH. Monomeric and dimeric LLT1 crystal structures originate from protein deglycosylated after the first GlcNAc, while the hexameric form corresponds to LLT1 with the original GlcNAc2Man5 glycosylation. The poster will present types of crystal interactions leading to formation of the four crystal structures. This study was supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, by the Czech Science Foundation (project 15-15181S), by the Ministry of Education, Youth and Sports of the Czech Republic (grant LG14009), by Charles University (UNCE 204025/2012, SVV 260079/2014, GA UK 161216), BioStruct-X (EC FP7 project 283570) and Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) supported by national member subscriptions. [1] Skálová et al., Acta Cryst., 2015, D71, 578-591. (Open access) [2] Bláha et al., Protein Expres. Purif. 2015, 109, 7-13. Figure 1. Structure of dimeric LLT1 with denoted positions of glycosylation sites.

Polymer and co-polymer surface modifying effects in the protein crystallization

Xylulose 5-phosphate (X5P) phosphoketolase (PK) is a central enzyme of the pentose phosphate pathway and in the same time, the largest representative of the thiamin diphosphate-dependent enzymes. In the presence of inorganic phosphate this enzyme converts X5P into glyceraldehyde 3-phosphate and acetyl phosphate. The ptk gene of PK from Lactococcus lactis was amplified by PCR and introduced into a prokaryotic expression vector. The enzyme was expressed as a fusion protein and purified by affinity and gel filtration chromatography. The purified protein thus obtained was electrophoretically homogeneous. The crystallization trials were performed using both hanging drop and sitting drop vapor diffusion techniques. Hanging drop procedure proved to be more efficient than sitting drop alternative procedure. Initially, crystal clusters were obtained; further optimization of protein purification as well as of crystallization conditions led to single crystals. X-ray diffractional tests on the crystals thus obtained evidenced that the crystals diffracted to approximately 3 Å. However the quality of the diffractional data still needs further improvement.