Crystallization of FrpC protein from Neisseria meningitidis

E. Tutubalina1, L. Bumba2, I. Kuta Smatanova1,3

¹University of South Bohemia in Ceske Budejovice, Faculty of Science, Branisovska 31, 370 05 Ceske Budejovice

²Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic

³Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady

Neisseria meningitidis is a Gram-negative bacterium that colonizes the nasopharynx of approximately 10% of population without causing any symptoms. However, the bacteria can penetrate across the nasopharyngeal mucosa, gain access to the bloodstream and pass the blood-brain barrier, inducing devastating invasive meningococcal diseases such as septicemia and/or meningitis.

Under conditions of limited iron availability, N. meningitides was shown to produce RTX1 (repeat in toxin) family proteins secreted through the type I pathway, the so-called FrpC-like proteins. These are characterized by the presence of a variable number of carboxyl-proximal glycine and aspartaterich repetitions of a nonapeptide RTX consensus motif (L/I/F)XGGXG(D/N)DX. But the biological activity of meningococcal FrpC-like proteins remains unknown.

It was reported that the FrpC protein undergoes a unique calcium-dependent autocatalytic processing at an Asp-Pro peptide bond that is accompanied by formation of high molecular weight oligomeric species of FrpC that contain subunits covalently cross-linked through a new type of isopeptide bond. The results strongly suggest that FrpC is also processed and cross-linked when meningococci grow in the calcium-rich environments of the mucosal secretions of nasopharynx and/or in the plasma and liquor during invasive infections.

One of the directions to investigate the function of FrpC protein is to study its structure. And the “clip-and-link” self-processing activity is one of the crucial aspects in purification step.

Initial crystallization trials of FrpC protein were performed using the sitting-drop vapour-diffusion at both 4^oC and 18 ^oC. The commercial screeening kits Morpheus, Structure, Pact Premier, MIDAS, PGA Screen and JCSG+ (Molecular Dimensions, Suffolk, England), PEG Suite (Qiagen sciences, Maryland, USA), Index Screen (Hampton Research, Aliso Viejo, USA), JCscreen Plus (Jena Bioscience, Jena, Germany) were tested to determine initial crystallization conditions using Gryphon high throughput crystallization robot (Art Robbins Instruments, USA).

This research is supported by the GACR P207/11/0717.