NEISSERIA MENINGITIDIS IRON-REGULATED PROTEIN FrpD: CRYSTALLIZATION AND CRYSTALLOGRAPHIC CHARACTERIZATION

Ekaterina Sviridova¹, Ladislav Bumba², Pavlina Rezacova^3,4, Katerina Prochazkova³, 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 37 Prague, Czech Republic

⁴Insitute of Molecular Genetics AS CR Flemingovo nam. 2, 166 37 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

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 = 2·10^-10 M) [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.

This project is aimed to determine the structure of FrpD protein. Our preliminary results showed 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 P6₂, 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. Structure refinement of the Se-Met FrpD_43-271 protein is currently in progress. The calculated structure will be further used as a search model in molecular replacement to determine the structure of native FrpD_43-271 protein.

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.

This work was supported by the Ministry of Education of the Czech Republic (LC06010 and MSM6007665808), by GA CR P207/11/0717 and by the Academy of Sciences of the Czech Republic (AV0Z60870520, AV0Z50520514 and AV0Z40550506).