MOLECULAR CHARACTERIZATION OF HYBRID TBP2 PROTEINS FROM NEISSERIA-MENINGITIDIS

Transferrin-binding protein 2 (Tbp2) from Neisseria is an outer membra ne-associated extracellular lipoprotein that is involved in iron captu re within the infected host. The analysis of tbp2 clones isolated from various bacterial strains revealed extensive divergences throughout t he open reading frame (ORF), with predicted amino acid (aa) sequences displaying 47% to 83% identity. Such a variability is likely to have r esulted from the selective pressure exerted by the host immune system, but raises questions regarding the existing constraints for conservat ion of protein function. Indeed, the neisserial Tbp2s include a large structured domain, extending throughout the N-terminal half of the pro tein (similar to 270-290 aa), which is extremely stable and whose conf ormational integrity is required for efficient binding to human transf errin (hTf). In this work, a functional study of Tbp2s encoded by hybr id genes constructed by reasserting highly divergent tbp2 sequences in the region of the ORF encoding this structured domain was performed. The data demonstrate that the determinant intramolecular interactions allowing formation of a stable Tbp2 structure able to interact efficie ntly with hTf or/and that the Tbp2 residues involved in the interactio n with hTf are not well conserved. However, a number of rearrangements appeared to generate genes encoding proteins which have retained stru ctural stability and hTf-binding capacity. This suggested that despite the extreme as sequence divergence and the conformational constraints , horizontal genetic exchanges, which are known to occur in neisserial populations, may have contributed significantly to the generation of sequence variation within tbp2 ORFs. The analysis of two tbp2 clones c haracterized in this work supports this hypothesis.