Abstract
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.
Highlights
The Gram-negative bacterium Neisseria meningitidis is a strictly human commensal that colonizes the nasopharynx of approximately 10% of healthy individuals
Adhesion of N. meningitidis to the mucosal epithelia of the human nasopharynx represents the first step of host colonization and subsequent development of meningococcal pathogenesis
Type IV pili are regarded as the major Neisseria adhesins, and rapid elongation of these polymeric filaments on the bacterial surface allows the initial attachment to the mucosal epithelia
Summary
The Gram-negative bacterium Neisseria meningitidis is a strictly human commensal that colonizes the nasopharynx of approximately 10% of healthy individuals. Tandemly repeated C-terminal RTX nonapeptides with the consensus motif GGxGxDxxx[14] These nonapeptides bind Ca2+ ions, and their sequential folding promotes secretion of RTX substrates from the Ca2+-free bacterial cytosol into the Ca2+-rich extracellular milieu through a dedicated Type I secretion system (T1SS)[15]. FrpC undergoes an autocatalytic and highly specific processing, which closely resembles a process known as “protein trans-splicing” (Fig. 1) This process includes Ca2+-induced autocatalytic cleavage of the Asp414-Pro[415] peptide bond and a subsequent covalent linkage of the released carboxyl group of Asp[414] of the FrpC1-414 fragment to an adjacent ε-amino group of another lysine residue through an isopeptide bond[18]. The trans-splicing activity of FrpC is mediated by a self-processing module (SPM), an adjacent segment of 177 residues (415–591), the primary amino acid sequence of which appears to be well-conserved in many RTX proteins from Gram-negative pathogens[19]. FrpD is translated as a 271-residue polypeptide, of which the first 24 residues constitute a type
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