CRYSTAL STRUCTURE OF THE IRON-REGULATED OUTER MEMBRANE LIPOPROTEIN FrpD (NMB0584) FROM NEISSERIA MENINGITIDIS

Ekaterina Sviridova^1,2, Ladislav Bumba³, Pavlina Rezacova^4,5, Peter Sebo^3,6, Ivana Kuta Smatanova^1,7

¹University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 37005 Ceske Budejovice

²University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and School of complex systems, Zamek 136, 373 33 Nove Hrady

³Academy of Sciences of the Czech Republic, Institute of Microbiology, Videnska 1083, 142 20 Prague

⁴Academy of Sciences of the Czech Republic, Institute of Organic Chemistry and Biochemistry, Flemingovo nam. 2, 166 10 Prague

⁵Academy of Sciences of the Czech Republic, Institute of Molecular Genetics, AS CR, Flemingovo nam. 2, 166 10 Prague

⁶Academy of Sciences of the Czech Republic, Institute of Biotechnology, AS CR, Videnska 1083, 142 20 Prague

⁷Academy of Sciences of the Czech Republic, Inst. of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady

Keywords: lipoprotein, iron-regulated protein

Introduction:

FrpD is a highly conserved lipoprotein of Neisseria meningitidis anchored to the bacterial outer membrane. The frpD gene sequence contains two translation initiation sites, which give rise to production of the full-length FrpD protein (FrpD₂₇₁) that harbours N-terminal signal peptide promoting FrpD export across the cytoplasmic membrane by Sec translocase, and the truncated FrpD protein (FrpD₂₅₀) that lacks the signal peptide and remaining in cytoplasm of the bacteria. The exported FrpD₂₇₁ precursor is processed to its mature form on the periplasmic side of the cytoplasmic membrane, sequentially modified by a lipid molecule at Cys₂₅ residue, and sorted to the outer bacterial membrane [1].

The biological function of FrpD appears to be linked to the FrpC protein, since FrpD was found to bind the N-terminal part of FrpC with very high affinity (K_d = 0.2 nM) [1]. However, mechanism of FrpD-FrpC interaction is unknown due to the absence of any structural information on these proteins. Moreover, the primary amino acid sequence of FrpD does not exhibit any similarity to known protein sequences of other organisms, and therefore, a new type of protein fold could be expected.

Results and discussion:

We found out that the full version of FrpD₂₅₀ protein couldn’t be crystallized. Therefore, we performed a specific truncation of 21 amino acid residues from N-terminus of FrpD₂₅₀ protein. The native and Se-Met substituted variants of recombinant, truncated version (lacking the first 21 amino acid residues from N-terminus) FrpD_43-271 protein were prepared and crystallized using the sitting-drop vapour-diffusion method. The crystals of native FrpD_43-271 protein belong to the hexagonal space group P6₄, while the crystals of Se-Met substituted FrpD_43-271 protein belong to the primitive orthorhombic space group P2₁2₁2₁ [2]. Crystal structure of Se-Met substituted FrpD_43-271 was determined using the single anomalous diffraction (SAD) method. The calculated structure was used as a search model in molecular replacement to determine the structure of native FrpD_43-271 protein. Here we present the crystal structures of the native and Se-Met derivative of the FrpD_43-271 (Se-Met FrpD_43-271) protein that were solved using diffraction data to 2.25 Å and 1.40 Å resolution, respectively.

References

1. K. Prochazkova, R. Osicka, I. Linhartova, P. Halada, M. Sulc, and P. Sebo, J. Biol. Chem., 280, (2005), 3251-3258.

2. E. Sviridova, L. Bumba, P. Rezacova, K. Prochazkova, D. Kavan, K. Bezouska, M. Kuty, P. Sebo and I. Kuta Smatanova, Acta Cryst. F, 66, (2010), 1119-1123.

Acknowledgements

This research was supported by the ME CR (COST LD11011, ME09016, CZ.1.05/2.1.00/01.0024), GACR (P207/12/0775, P207/11/0717), by the AS CR (AV0Z60870520) and GAJU 170/2010/P for ES.