Crystallization study of
the Iron-regulated outer membrane lipoprotein (FrpD)
from Neisseria meningitidis
E. Sviridova1,
L. Bumba2, P. Sebo,3 and I. Kuta Smatanova1,4
1Institute of Physical Biology USB CB, Zamek
136, 373 33 Nove Hrady,
Czech Republic
2Institute of Microbiology AS CR, Videnska
1083, 142 20 Prague, Czech Republic
3Institute of Biotechnology AS CR, Videnska
1083, 142 20 Prague, Czech Republic
4Institute of Systems Biology and Ecology AS CR, Zamek 136, 373 33 Nove Hrady, Czech Republic
sviridova@greentech.cz
Neisseria meningitidis
is a Gram-negative
bacterium colonizing the nasopharynx of about 10% of
healthy humans. Occasionally the meningococci
can traverse the mucosal epithelia to reach the bloodstream, eventually
cross the blood-brain barrier, and cause rapidly progressing septicemia and/or
meningitis [1]. Several traits potentially
required for virulence of meningococci have been
identified, including production of a capsule conferring resistance to serum,
secretion of an IgA protease, the high antigenic
variability of pili and non-fimbrial
adhesins, and the presence of several iron
acquisition systems [2]. Under conditions of limited iron availability, N. meningitidis
produces Fe-regulated proteins, FrpD and FrpC. FrpC belongs to a family of
type I-secreted RTX (Repeat in toxins) proteins and it may be involved in the
pathogenesis of meningococcal
infection. FrpD binds the N-terminal portion of FrpC with a very high
affinity and probably serves as an accessory lipoprotein involved in anchoring
of the secreted RTX protein to the outer bacterial membrane [3]. The aim of
this project is to produce crystals of FrpD protein
for X-ray diffraction experiments and to solve the structure of FrpD protein.
The
recombinant, truncated version of the FrpD protein
lacking the first 21 amino acid residues (FrpD250) with the
C-terminal polyhistidine tag,
was expressed in E. coli BL21λDE3
and purified using a combination of metal affinity and anion-exchange column
chromatography. The crystals of truncated FrpD
protein lacking the first 42 amino acid residues were obtained using a sitting
drop vapour diffusion method. Diffraction data were
collected at the beamline MX
BL14.1 of synchrotron BESSY (Berlin, Germany) at 100 K to the resolution
of 2.27 A0.
Crystals
of FrpD belong to the hexagonal space group P 6 2,
with unit-cell parameters
a = b = 115.33 Å, c =
38.79 Å and α = β = 90° and γ = 120°. To determine the structure of the FrpD protein, phase problem has to be solved using
single/multiple anomalous diffraction (SAD/MAD) experiment hence the
crystallization of selenomethionine derivative FrpD protein is currently in progress.
References
1. M. Guibourdenche, E. A. Hoiby, J. Y.
Riou, F. Varaine, C. Joguet and D. A Caugant,
Epidemiology and Infection, 116, (1996), 115-120.
2. Y. L.Tzeng, D. S. Stephens, Microbes and Infection, 2, (2000), 687–700.
3. K. Prochazkova, R. Osicka, I. Linhartova, P. Halada, M. Sulc, and P. Sebo, The Journal of
Biological Chemistry, 280,
(2005), 3251-3258.
This project was supported by grants MSM6007665808 and LC06010
(Ministry of Education of the Czech Republic), AVOZ60870520 (Academy of
Sciences of the Czech Republic) and GACR 310/06/0720