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.

 

Acknowledgements.

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