CRYSTALLIZATION STUDY OF THE IRON-REGULATED OUTER MEMBRANE LIPOPROTEIN (FrpD) FROM NEISSERIA MENINGITIDIS

Ekaterina Sviridova¹, Ladislav Bumba³, Peter Sebo^3,4, Ivana Kuta Smatanova^1,2

¹Institute of Physical Biology USB CB, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Institute of Systems Biology and Ecology AS CR, Zamek 136, 373 33 Nove Hrady, Czech Republic

³Institute of Microbiology AS CR, Videnska 1083, 142 20 Prague, Czech Republic

⁴Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague, Czech Republic

Keywords: biocrystallization, lipoprotein, iron-regulated protein

Introduction:

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. The molecular basis of meningococcal virulence remains difficult to analyze, because human colonization and invasive disease are not adequately reproduced in current animal models. Several traits potentially required for virulence of meningococci have, however, 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.

Under conditions of limited iron availability, N. meningitidis produces Fe-regulated proteins, FrpD and FrpC, which both are encoded consecutively in an iron-regulated frpDC operon controlled by a ferric uptake regulator (Fur). 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 due to the presence of high titers of anti-FrpC antibodies in convalescent-phase sera of a number of patients after invasive meningococcal disease. FrpD is synthesized with a type II signal peptide for export across the cytoplasmic membrane. It is posttranslationaly modified by a lipid molecule and is targeted to the outer bacterial membrane. FrpD is highly conserved in meningococcal strains and its primary amino acid sequence does not exhibit any similarity to any known protein sequences of other organisms. Furthermore, FrpD binds the N-terminal portion of FrpC (first 300 residues) with very high affinity (apparent K_d=0.2 nM) and probably serves as an accessory lipoprotein involved in anchoring of the secreted RTX protein to the outer bacterial membrane.

Results and discussion:

The aim of this project is to produce crystals of FrpD protein for X-ray diffraction experiments and to solve the structural features 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 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 Ǻ. 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 = b = 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 preparation of selenomethionine labeling and/or heavy atom derivatives are currently in progress.

References:

M. Guibourdenche, E. A. Hoiby, J. Y. Riou, F. Varaine, C. Joguet and D. A Caugant, Epidemiology and Infection, 116, (1996), 115-120.

Y. L.Tzeng, D. S. Stephens, Microbes and Infection, 2, (2000), 687–700.

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.