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

 

Karla Fejfarová1*, Petr Man2, Hynek Mrázek2, Alan Kádek2,3, Jarmila Dušková4, Tereza Skálová4,  Petr Kolenko1, Tomáš Kovaľ1, Jan Stránský4,5, Jindřich Hašek4, Jan Dohnálek1,4


1Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2/1888, 162 06 Prague 6, Czech Republic

2Institute of Microbiology AS CR, v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic
3Faculty of Science, Charles University in Prague,
Albertov 6, 128 43 Prague 2, Czech Republic
4Institute of Biotechnology AS CR, v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic
5Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7, 115 19 Prague 1, the Czech Republic

* correspondence e-mail: fejfarova@imc.cas.cz

 

Carnivorous pitcher plants of the genus Nepenthes secrete their own aspartic proteases, nepenthesins, to digest prey. Nepenthesins differ significantly in sequence from other plant aspartic proteases. This difference, which brings more cysteine residues into the structure of nepenthesins, in conjunction with putative N-glycosylation, can contribute to uniquely high temperature and pH stabilities of these proteases [1, 2].

In continuation of our previous study of the expression and biochemical and enzymatic characterization of a recombinant form of nepenthesin-1 (rNep-1) from Nepenthes gracilis [3], we report its crystallization and preliminary X-ray analysis. Crystals of rNep-1 in complex with the pepstatin A inhibitor have been grown using the hanging-drop vapour-diffusion technique. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at Bessy II of HZB, Berlin. The crystals belong to space group P21, with unit-cell parameters a = 54.4 Å, b = 86.6 Å, c = 95.8 Å, β = 106°. The self-rotation function combined with solvent-content calculations and Matthews coefficient suggest presence of two molecules of rNep-1 in the asymmetric unit.

This work was supported by the Ministry of Education, Youth and Sports
of the Czech Republic (
grants No. EE2.3.30.0029 and No. LG14009), by BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund, and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14.

[1] K. Takahashi, S. B. P. Athauda, K. Matsumoto et al., Curr. Protein Pept. Sci., 2005, 6, 513–525.

[2] S. B. P. Athauda, K. Matsumoto, S. Rajapakshe et al., Biochem. J., 2004, 381, 295–306.

[3] A. Kadek, V. Tretyachenko, H. Mrazek et al., Protein Expr. Purif., 2014, 95, 121–128.