The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization

Abstract

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.

Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

“…The contacts present between the domains could somewhat restrict interdomain flexibility, which is in line with that observed for RrgB, whose four domains also display limited structural malleability (24). Two of RrgC domains are IgG-like; their potential function could be to enhance the binding capacity of RrgC (and hence the pilus) to the cell wall or to eukaryotic targets.…”

“…In S. pneumoniae, the pilus backbone is formed by covalently associated RrgB units; microscopy efforts have indicated that the molecules are associated with a directionality, in which a noselike protrusion defines the polarity of the fiber (25). The crystal structure of RrgB, fitted into a cryo-EM map of the pilus, confirms this polarity (24,59). Notably, RrgA and RrgC were suggested as being located at the extremities of the fiber, with RrgC at the base and RrgA at the free end (25).…”

“…Isopeptide bonds have now been identified in a number of pilin subunits from Gram-positive bacteria, including RrgA and RrgB, which display such bonds in almost all domains (24,26,59). In RrgC, the isopeptide bonds of domains D2 and D3 had been identified through sequence comparisons and mutagenesis studies; the latter indicated that both bonds play a key role in protein stability (22).…”

“…Notably, RrgA and RrgC were suggested as being located at the extremities of the fiber, with RrgC at the base and RrgA at the free end (25). The structure of RrgA revealed a four-domain flexible molecule carrying a von Willebrand factor (VWF) domain capable of recognizing extracellular matrix components (26), whereas RrgB displayed a threedomain fold (24,59). Until recently, however, the structure of the third structural component of the pneumococcal pilus, RrgC, was still lacking, impeding a more complete understanding of the architecture of the fiber from S. pneumoniae.…”

“…The backbone is formed by the covalent association of RrgB monomers (Fig. 6, bright green), and its nucleophilic Lys-183 residue thus has a dual recognition potential, participating in the covalent association with RrgA-YPRTG and to the IPQTG motif of other RrgB molecules to generate the fiber with the adhesin at its tip (21,24,25,59). Finally, RrgC (Fig.…”