The iron-regulated protein FrpD from Neisseria meningitidis is an outer membrane lipoprotein that interacts with very high affinity (Kd ~ 0.2 nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the Repeat in ToXin (RTX) protein family. In the presence of Ca2+, FrpC undergoes Ca2+ -dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp414-Pro415 peptide bond and formation of an Asp414-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC1-414. We identified FrpD residues involved in FrpC1-414 binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC1-414 complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC1-414 fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC1-414 complex may be involved in the interaction of meningococci with the host cell surface.

Center for Nanobiology and Structural Biology Institute of Microbiology Czech Academy of Sciences Zamek 136 37333 Nove Hrady Czech Republic

EMBL Hamburg Outstation c o DESY Notkestrasse 85 D 22603 Hamburg Germany

Faculty of Science University of South Bohemia Ceske Budejovice Branisovska 1760 37005 Ceske Budejovice Czech Republic

Institute of Microbiology Czech Academy of Sciences Videnska 1083 14220 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 2 16610 Prague Czech Republic

Zobrazit více v PubMed

Pizza M. & Rappuoli R. PubMed

Rosenstein N. E., Perkins B. A., Stephens D. S., Popovic T. & Hughes J. M. Meningococcal disease. N. Engl. J. Med. 344, 1378–1388 (2001). PubMed

Rouphael N. G. & Stephens D. S. PubMed PMC

Stephens D. S., Greenwood B. & Brandtzaeg P. Epidemic meningitis, meningococcaemia, and PubMed

Hill D. J., Griffiths N. J., Borodina E. & Virji M. Cellular and molecular biology of PubMed PMC

Capecchi B. et al. PubMed

Mattick J. S. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56, 289–314 (2002). PubMed

Sadarangani M., Pollard A. J. & Gray-Owen S. D. Opa proteins and CEACAMs: pathways of immune engagement for pathogenic PubMed

Scarselli M. et al. PubMed

Grifantini R. et al. Identification of iron-activated and –repressed Fur-dependent genes by transcriptome analysis of PubMed PMC

Basler M. et al. The iron-regulated transcriptome and proteome of PubMed

Thompson S. A., Wang L. L., West A. & Sparling P. F. PubMed PMC

Osicka R., Kalmusova J., Krizova P. & Sebo P. PubMed PMC

Linhartova I. et al. RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol. Rev. 34, 1076–1112 (2010). PubMed PMC

Bumba L. et al. Calcium-driven folding of RTX domain β-rolls ratchets translocation of RTX proteins through Type I secretion ducts. Mol. Cell 62, 47–62 (2016). PubMed

Forman S. et al. PubMed PMC

Linhartova I. et al. Meningococcal adhesion suppresses proapoptotic gene expression and promotes expression of genes supporting early embryonic and cytoprotective signaling of human endothelial cells. FEMS Microbiol Lett. 263, 109–118 (2006). PubMed

Osicka R. et al. A novel “Clip-and-link” activity of repeat in toxin (RTX) proteins from Gram-negative pathogens. J. Biol. Chem. 279, 24944–24956 (2004). PubMed

Matyska Liskova P. et al. Probing the Ca PubMed

Kuban V., Novacek J., Bumba L. & Zidek L. NMR assignment of intrinsically disordered self-processing module of the FrpC protein of PubMed

Sadilkova L. et al. Single-step affinity purification of recombinant proteins using a self-excising module from PubMed PMC

Prochazkova K. et al. The PubMed

Kovacs-Simon A., Titball R. W. & Michell S. L. Lipoproteins of bacterial pathogens. Infect. Immun. 79, 548–561 (2011). PubMed PMC

Sviridova E. et al. Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from PubMed PMC

Ezezika O. C. et al. Incompatibility with formin Cdc12p prevents human profilin from substituting for fission yeast profiling: insights from crystal structures of fission yeast profiling. J. Biol. Chem. 284, 2088–2097 (2009). PubMed PMC

Prlic A. et al. Precalculated protein structure alignments at the RCSB PDB website. Bioinformatics 26, 2983–2985 (2010). PubMed PMC

Shindyalov I. N. & Bourne P. E. Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. Protein Eng. 11, 739–747 (1998). PubMed

Bumba L., Sviridova E., Kuta Smatanova I., Rezacova P. & Veverka V. Backbone resonance assignments of the outer membrane lipoprotein FrpD from PubMed

Williamson M. P. Using chemical shift perturbation to characterise ligand binding. Prog. Nucl. Magn. Reson. Spectrosc. 7, 1–16 (2013). PubMed

Muller D. R. et al. Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927–1934 (2001). PubMed

Young M. M. et al. High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry. Proc. Natl. Acad. Sci. USA 97, 5802–5806 (2000). PubMed PMC

Bakkes P. J., Jenewein S., Smits S. H., Holland I. B. & Schmitt L. The rate of folding dictates substrate secretion by the PubMed PMC

Staab J. F., Bradway S. D., Fidel P. L. & Sundstrom P. Adhesive and mammalian transglutaminase substrate properties of PubMed

Sutherland T. C., Quattroni P., Exley R. M. & Tang C. M. Transcellular passage of PubMed PMC

Schaller A. et al. Characterization of apxIVA, a new RTX determinant of PubMed

Liu J. et al. PubMed

Mueller U. et al. Facilities for macromolecular crystallography at Helmholtz-Zentrum Berlin. J. Synchrotron Radiat. 19, 442–449 (2012). PubMed PMC

Minor W., Cymborowski M., Otwinowski Z. & Chruszcz M. HKL-3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859866 (2006). PubMed

Perrakis A., Morris R. & Lamzin V. S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999). PubMed

Cowtan K. Error estimation and bias correction in phase-improvement calculations. Acta Crystallogr. D Biol. Crystallogr. 55, 1555–1567 (1999). PubMed

Vagin A. & Teplyakov A. An approach to multi-copy search in molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 56, 1622–1624 (2000). PubMed

Murshudov G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011). PubMed PMC

Emsley P. & Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). PubMed

Chen V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010). PubMed PMC

Krieger E., Koraimann G. & Vriend G. Increasing the precision of comparative models with YASARA NOVA-a self-parameterizing force field. Proteins 47, 393–402 (2002). PubMed

Holm L. & Sandler C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993). PubMed

Orengo C. et al. CATH – a hierarchic classification of protein domain structures. Structure 5, 1093–1108 (1997). PubMed

Krissinel E. & Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004). PubMed

Blanchet C. E. et al. Instrumental setup for high throughput solution scattering at the X33 beamline of EMBL-Hamburg. J. Appl. Cryst. 45, 489–495 (2012).

Franke D. & Svergun D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J. Appl. Cryst. 42, 342–346 (2009). PubMed PMC

Kukacka Z., Rosulek M., Strohalm M., Kavan D. & Novak P. Mapping protein structural changes by quantitative cross-linking. Methods 89, 112–20 (2015). PubMed

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins