Karla Fejfarová

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

The single–strand–specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1–P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X–ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1–P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes–shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1–P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

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.

Highly stable single-strand-specific 3′-nuclease/nucleotidase from Legionella pneumophila

The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.

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 2.8u2005Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40u2005Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78u2005Å, 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.

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

Zinc dependent 3'-nucleases/nucleotidases from plants, fungi and bacteria – varied roles and specificity

S1-P1 3-nucleases/nucleotidases (EC 3.1.30.1) are small, mostly alpha-helical enzymes relying on the active centre formed by three zinc ions and surrounded by varied nucleotide binding sites [1]. These extracellular enzymes are employed in various roles in plants, fungi and bacteria but typically not in mammals. These enzymes are utilized in plants in apoptotic processes, tissue development and senescence, in protozoan parasites for securing nutrients and a similar role is expected in some gram-negative bacteria. The purpose of the enzyme in a given organism seems to lead to modification of substrate specificity. Tomato TBN1 has almost universal ability to cleave nucleic acids – single strand, double strand DNA, RNA and structured RNA and this is reflected by the appearance of the enzyme surface [2, 3]. Fungal S1 nuclease shows basically strict ssDNA preference. The enzyme specificity is defined by the width of the cleft, presence of nucleotide binding sites and distribution of electrostatic potential. We have determined a series of complexes with a fungal enzyme showing a range of ligand-protein interactions. The required enzyme specificity/substrate promiscuity is connected with particular arrangement of surface sites. S1-P1 nuclease is also present in gram negative bacteria and protozoan parasites pathogenic to humans [e.g. 4]. We have cloned, expressed and purified nuclease from a human pathogen and proved its activity. Recent results leading aimed at clarification of specificity features, stabilization of the enzyme in bacteria and inhibition potential, will be presented. The project is supported by MEYS CR (LG14009, LQ1604 NPU II), CZ.1.05/1.1.00/02.0109 BIOCEV provided by ERDF and MEYS, and CSF (15-05228S). References 1. Romier C., Dominguez R., Lahm A., et al. Proteins 1998, vol. 32, no. 4, 414–424. 2. Koval T., Lipovová P., Podzimek T., et al. Acta Cryst. 2013, vol. D69, no. 2, 213-226. 3. Stránský J., Koval T., Podzimek T., et al. Acta Cryst. 2015, vol. F71, 1408-1415. 4. Guimarães-Costa A.B., DeSouza-Vieira T.S., Paletta-Silva R., et al. Infect. Immun. 2014, vol. 82, no. 4, 1732-40. Figure 1. Complex of TBN1 and phosphate ion: mimic of product binding mode (PDB ID 4JDG).

Optimization in S-SAD phasing - difference between solved and unsolved structure

J. Stránský, T. Kovaľ, L. Østergaard, J. Dušková, T. Skálová, J. Hašek, P. Kolenko, K. Fejfarová, J. Dohnálek Academy of Sciences of the Czech Republic, Institute of Biotechnology, Prague, Czech Republic, Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic, Academy of the Sciences of the Czech Republic, Institute of Macromolecular Chemistry, Prague , Czech Republic, Novozymes A/S, Bagsvaerd, Denmark

Crystallization and preliminary X-ray diffraction analysis of nepenthesin-1

1 Institute of Macromolecular Chemistry AS CR, v.v.i., Prague, Czech Republic, 2 Institute of Microbiology AS CR, v.v.i., Prague, Czech Republic, 3 Charles University in Prague, Faculty of Science, Prague, Czech Republic, 4 Institute of Biotechnology AS CR, v.v.i., Prague, Czech Republic, 5 Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Prague, Czech Republic