Sortase-assembled pili in Corynebacterium diphtheriae are built using a latch mechanism

Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine–isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae. We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.

The cellular surface of many bacteria is elaborated with thin appendages called pili (also called fimbriae) which have a range of roles including twitching motility, conjugation, immunomodulation, biofilm formation, and adherence (1, 2). These long proteinaceous fibers are key virulence factors that mediate initial host–pathogen interactions, which are subsequently strengthened by more intimate contacts from shorter pili and cell wall–attached adhesins (1–13 ). As the infection progresses, pili also facilitate biofilm formation, protecting invading microbes from host immune clearance and exogenous antibiotics (1, 5, 11, 14). Gram-positive bacteria display very thin (1 to 5 nm) (15) hair-like pili that nevertheless possess enormous tensile strength because their protein components are crosslinked together by lysine–isopeptide bonds. These crosslinked fibers are displayed by a wide range of pathogenic and commensal Gram-positive bacteria, but their structures and mechanism of assembly remain poorly understood (2, 5, 7–10).Pili in Gram-positive bacteria are assembled by pilus-specific sortase enzymes that crosslink the pilus subunits (called pilins) together via lysine–isopeptide bonds. Our current understanding of this process has been significantly advanced by studies of the SpaA pilus in Corynebacterium diphtheriae, a pathogen that causes pharyngeal diphtheria (5, 8, 10, 16). The SpaA pilus mediates adherence to the pharyngeal epithelium and consists of three types of pilins: the pilus shaft is formed by SpaA, and the tip and base are formed by SpaC and SpaB, respectively (17). The C. diphtheriae pilus-specific sortase (^CdSrtA) assembles the pilus by catalyzing a repetitive, irreversible transpeptidation reaction that covalently links pilin subunits together via an isopeptide bond. The shaft of the pilus is formed by ∼100 to 250 crosslinked SpaA pilins (16). ^CdSrtA-catalyzed SpaA polymerization begins when SpaA prepilin proteins containing an N-terminal signal peptide sequence are exported via the Sec pathway and retained on the extracellular surface via a C-terminal cell wall sorting signal (CWSS) (18). ^CdSrtA then crosslinks SpaA proteins together via a two-step process. First, an LPLTG sorting signal sequence within the CWSS is cleaved between Thr and Gly residues by the sortase, generating a thioacyl-linked ^CdSrtA-SpaA intermediate in which the enzyme’s active site cysteine residue is covalently linked to the carbonyl atom of the sorting signal threonine. In the second step, a lysine ε-amine group originating from another SpaA pilin attacks the thioacyl linkage in the ^CdSrtA-SpaA intermediate, thereby joining distinct SpaA proteins together via a K190-T494 isopeptide bond (Fig. 1A). The reactive lysine in SpaA is housed within the N-terminal domain and is part of a highly conserved WxxxVxVYPK sequence motif that is found in many pilin proteins (17). The shaft of the pilus is constructed by repeating this two-step process, and a similar ^CdSrtA-catalyzed reaction is used to add the SpaC tip pilin to SpaA. Pilus assembly is completed by incorporating the SpaB basal pilin, which promotes pilus attachment to the cell wall using a distinct housekeeping sortase ^CdSrtF (3). Pilus biogenesis is thought to occur within “pilusosomes” on the cell surface, at which pilin substrates and pilus-specific sortases colocalize to facilitate rapid polymerization (18).Open in a separate windowFig. 1.Structure of the ^NSpaA-signal peptide complex. (A) Schematic of pilus polymerization with full-length SpaA molecules. An expanded view of the two portions of the crosslinked SpaA polymer investigated in this study, ^CSpaA-^NSpaA complex and ^NSpaA-signal, are boxed in gray dashed lines and solid black lines, respectively. (B) The ^NSpaA-signal peptide complex is represented in surface representation with relative conservation of each residue indicated by a color gradient ranging from highly variable positions (blue) to highly conserved residues (yellow). The peptide (magenta sticks) is docked into a highly conserved, nonpolar binding groove on SpaA. (C) A bundle of the 40 lowest energy structures of the SpaA-signal complex is displayed. The backbone of the ^NSpaA domain is represented by blue ribbons. The last five residues of the sorting signal peptide are depicted as red sticks, and Lys190 is shown as green sticks. (D) Secondary structural elements of the NMR structure are highlighted. (E) An expanded view of the peptide binding interface shows how the peptide is bound in the cleft of ^NSpaA. Residues on SpaA exhibiting intermolecular NOEs to the peptide are shown as sticks. Interacting residues in the core of the domain and within the AB loop are colored yellow and pink, respectively.Despite their importance in bacterial physiology and pathogenesis, only structures of isolated, noncrosslinked pilins have been determined at atomic-level resolution (19). This is because it has been challenging to obtain homogenous crosslinked pili that are suitable for biophysical analyses and because Gram-positive pili are thin and flexible, making them difficult to study using cryogenic electron microscopy and X-ray crystallography. Crystal structures of isolated pilins have revealed that they contain IgG-like Cna-type domains and frequently one or more spontaneously forming intradomain isopeptide bonds that impart significant resistance to mechanical forces (19–21). Internal isopeptide bond linkages exist as either D- or E-type and are extremely stabilizing, allowing pilin domains to withstand the highest unfolding forces yet reported for a globular protein (20). Atomic-level structures of sortase crosslinked pilins have yet to be visualized, but a transmission electron microscopy study of the Streptococcus pneumoniae RrgB pilus enabled the periodicity and polarity of individual subunits within the pilus fiber to be determined (22). This work revealed that the subunits in the pilus are arranged in a head-to-tail manner, enabling sortase-catalyzed isopeptide crosslinking between the lysine and LPxTG motifs located at the N- and C-terminal ends of the pilin, respectively. In crystals, similar head-to-tail packing arrangements are observed, but whether these lattice interactions are also present in the intact pilus is not known.In this study, we used a recently developed in vitro pilus assembly system and hybrid structural biology methods to gain insight into the structure and biogenesis mechanism of the SpaA pilus from C. diphtheriae. We first determined the NMR structure of the N-terminal domain of SpaA crosslinked to the sorting signal peptide and then used small angle X-ray scattering (SAXS), NMR, and crystallographic data to model the structure of the isopeptide-linked SpaA-SpaA building block that is repeated to construct the pilus shaft. We show that crosslinking is accompanied by a large disordered-to-ordered structural change in the SpaA pilin, which forms an interpilin interface that differs markedly from packing interactions observed in crystals of the isolated SpaA. Quantitative measurements of kinetics of the sortase-catalyzed transpeptidation reaction suggest that the enzyme uses a latch mechanism to select the appropriate lysine residue on SpaA for interpilin crosslinking.