The NMR structure of the self-processing module (SPM) of the Neisseria meningitidis FrpC protein. (A) Schematic representation of the Ca2+-dependent clip-and-link of FrpC. The Ca2+-induced folding of FrpC is associated with a Ca2+-dependent conformational switch of SPM (residues 415 to 591 of FrpC, in orange), which promotes autocatalytic processing of the D414-P415 peptide bond and covalent linkage of the released D414 residue to an e-amino group of a neighboring lysine residue through an Asp-Lys isopeptide bond. The residues of the putative EF-hand-like Ca2+-binding motifs are underlined. (B) Overlay of the 1H-15N HSQC spectra of 15N-labeled SPM in the absence (-) and in the presence (+) of 10 mM CaCl2. (C) Overlay of backbone traces of the 20 lowest energy structures of Ca-SPM solved by NMR, shown in a rainbow representation from blue (N terminus) to red (C terminus). (D) Topology of secondary structure elements of Ca-SPM generated by Pro-origami.
The posttranslational Ca2+-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free e-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neiseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.
Kuban, V.; Macek, P.; Hritz, J.; Nechvatalova, K.; Nedbalcova, K.; Faldyna, M.; Zidek, L. & Bumba, L.: Structural Basis of Ca2-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins, mBio. 2020, 11:e00226-20, https://doi.org/10.1128/mBio.00226-20.