CRYSTALLIZATION STUDY OF THE IRON-REGULATED PROTEIN FrpD FROM NEISSERIA MENINGITIDIS

Ekaterina Sviridova¹, Ladislav Bumba², Pavlina Rezacova^3,4, Peter Sebo^2,5, Ivana Kuta Smatanova^1,6

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 Organic Chemistry and Biochemistry, Flemingovo nam. 2, 166 10 Prague, Czech Republic

⁴Insitute of Molecular Genetics AS CR Flemingovo nam. 2, 166 10 Prague, Czech Republic

⁵Institute of Biotechnology AS CR, Videnska 1083, 142 20 Prague, Czech Republic

⁶Institute of Systems Biology and Ecology AS CR, Zamek 136, 373 33 Nove Hrady, Czech Republic

Keywords: lipoprotein, iron-regulated protein

Introduction:

Neisseria meningitidis is a highly diverse facultative bacterial pathogen. In most instances it colonizes its human host without causing disease. Its occasional invasion, however, can entail devastating diseases like septicemia or meningitis. Molecular basis of meningococcal virulence remains difficult to analyze. However, several traits potentially required for virulence of meningococci have been identified, including 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.

The iron-regulated protein FrpD was identified as a N. meningitidis outer membrane lipoprotein. FrpD is highly conserved in a set of meningococcal strains and its primary amino acid sequence does not exhibit any similarity to known protein sequences of other organisms. The frpdD gene has the potential to encode two protein variants the 271-residue long FrpD sequence (FrpD₂₇₁) and the 250-residue short sequence (FrpD₂₅₀)_. FrpD₂₇₁ is synthesized with a type II signal peptide for export across the cytoplasmic membrane. Then it is posttranslationally modified by a lipid molecule and targeted to the outer bacterial membrane. FrpD₂₅₀ lacks the signal peptide and possibly remains in the cytoplasm. The exported lipidated FrpD, as well as its recombinant non-exported FrpD₂₅₀ protein are both able to bind the N-terminal portion of FrpC (within first 300 residues) with very high affinity (apparent K_d=0.2 nM). FrpD may serve as an accessory lipoprotein involved in anchoring of the secreted FrpC protein to the outer bacterial membrane.

Results and discussion:

This project is aimed to determine the structure of FrpD protein and to perform the structural characterization of FrpD. Our preliminary results showed the full version of FrpD₂₅₀ protein can not be crystallized. Therefore, we performed a specific truncation of 21 amino acid residues from N-terminus of FrpD₂₅₀ protein. The recombinant, truncated version (lacking the first 21 amino acid residues from N-terminus) FrpD₂₅₀ protein was expressed in non-methionine auxotrophic Escherichia coli BL21λDE3 cultivated on the media containing selenomethionine (Se-Met). Se-Met derived FrpD protein was purified using a combination of metal affinity and gel-filtration 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 Ǻ. The diffraction data will be used to determine the structure of FrpD by single/multiple anomalous diffraction (SAD/MAD) method.

References:

N.E. Rosenshtein, B.A. Perkins, D.S. Stephens, T. Popovic, and J.M. Hughes, The New England Journal of Medicine, 344, (2001), 1378-1388.

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.