(a) Schematic representation showing the predicted domain organization of GG-SpaA. GG-SpaA consists of two CnaB-type domains, with regions for the secretion and LPXTG motif sorting signals at the N- and C-terminals, respectively. Arrows indicate positions of the pilin motif and E-box. Residue locations for the lysine-asparagine (K-N) and lysine-aspartate (K-D) isopeptide bonds are shown. (b) Ribbon cartoon of the GG-SpaA crystal structure. GG-SpaA consists of two Ig-like domains, with each having a CnaB-type fold. N- and C-terminally located domains are labeled as N-domain and C-domain, respectively. Core β-strands forming the β-sandwich fold are rainbow colored (red to violet) according to the topology diagram (see Fig. 1c). Residues forming isopeptide bonds are shown in sticks. The essential lysine (K171) of the pilin motif at the domain interface and the leucine (L301) and proline (P302) residues of the C-terminally located LPXTG motif are also shown in sticks. Extended loop regions are labeled accordingly. Major β-strands unrelated to the core CnaB fold are shown in cyan. (c) Topology diagram depicting the secondary structure of the GG-SpaA CnaB fold. Core β-strands of the β-sandwich fold are sequentially labeled (A to G) starting from the N- to C-terminals and rainbow colored (red to violet). An isopeptide bond between β-strands A and G is indicated by a red horizontal line.

(a) The isopeptide bond in the N-domain forms between K47 and N172, and also involves the catalytic glutamate at position 139. Residues for isopeptide bond formation (K47, N172, and E139), adjacent hydrophobic residues, and the essential lysine of the pilin motif (K171) are shown in sticks. The electron density (2Fo-Fc) map is contoured at 1.5 σ. (b) Isopeptide bond formation in the C-domain involves K184, D295, and catalytic E269. Residues for the isopeptide bond and nearby hydrophobic residues are shown in sticks. The electron density (2Fo-Fc) map is contoured at 1.5σ. (c) Superposition of the N- and C-domains (gold and cyan, respectively) is depicted. Location of isopeptide bonds is indicated by an arrow. Neighboring hydrophobic residues are shown in sticks. Major loop perturbations are labeled (AB, BC, and DE).

(a) Four copies of the C-domain GG-SpaA are in the asymmetric unit of the orthorhombic crystal form. Core β-strands of the β-sandwich are rainbow colored as in . Metal ions are depicted as gray spheres. (b) Enlarged view of one metal ion coordinated by aspartates from self (D283) and two crystal symmetry mates (D204* and D277*). (c) N-domain of GG-SpaA obtained from E269A mutant protein. An isopeptide bond is not formed between K47 and N172, despite the presence of a glutamate at position 139 in the N-domain. While K47 is engaged in hydrogen bonding with E139, N172 is pointing away from K47. The core β-sandwich is rainbow colored as in , but the loops and critical residues (sticks) are shown in pink for highlighting their conformational changes in comparison with that of full-length WT (in gray and green). Major changes in the AB and EF loops are indicated by an oval (dashed lines) in pink and blue, respectively. Movement of the C-terminal linker region (residues 170–180) is shown by an arrow (black).

(a) E139A mutant. Shown is the absence of the isopeptide bond between K47 and N172 when an alanine is at position 139 in the N-domain. The electron density (2Fo-Fc) map is contoured at 1.5σ. (b) E269A mutant. An isopeptide bond is formed between K184 and D295 when an alanine is at position 269 in the C-domain. The electron density (2Fo-Fc) map is contoured at 1.5σ. (c) E269A mutant. Difference electron density map contoured at 3σ for the isopeptide bond in the E269A mutant. K184 and D295 were mutated to Ala, and E269 was kept intact in the electron density (Fo-Fc) map calculation. The positive and negative densities are shown in green and red, respectively. (d) E269A mutant. An omit map calculated using Sfcheck in the CCP4 suite is contoured at 1.5σ and shows continuous electron density for isopeptide bond between K184 and D295 when an alanine is at position 269 in the C-domain. (e) D295N mutant. Shown is isopeptide bond formation between K184 and N295 with glutamate at position 269 in the C-domain. The electron density (2Fo-Fc) map is contoured at 1.5σ. (f) D295N/E269A mutant. An omit map calculated using Sfcheck in the CCP4 suite is contoured at 1.5σ and shows continuous electron density for isopeptide bond between K184 and N295 when an alanine is at position 269 in the C-domain.

(a) Proteolytic stability of WT and mutant GG-SpaA. Trypsinized GG-SpaA proteins (WT and the E139A, D295N, E269A (E1) and E269A (E2)) were sampled at various time points (0, 1, 3, and 24 h) and analyzed by SDS-PAGE. Molecular weight markers (kDa) are indicated on the left. For clarity, the lanes were cropped from different gels (), which have been run under the same experimental conditions. (b) Circular dichroism (CD) spectra of WT and mutant GG-SpaA measured at 20 °C over the wavelength range of 200–260 nm. The spectra are colored as follows, WT (blue), D295N (orange), E139A (red), E269A (green), E139A/E269A (violet), and D295N/E269A (cyan). (c) Thermal denaturation curves for WT and mutant GG-SpaA. Calculation of thermal stability was based on the change in molar ellipticity at 220 nm as a function of the increased temperature. The 220 nm wavelength near maxima was chosen for making the calculations since there is significant signal noise around the minimum at 207 nm. Curves are colored as in Fig. 5b. (d) Recombinant lactococcal cells were nisin induced and analyzed for SpaCBA-pilus production by immunoblotting with anti-GG-SpaA serum. Included are the empty vector GRS1052 (lane 1), WT SpaCBA-piliated GRS1185 (lane 2), E139A-substituted GRS1213 (lane 3), E269A-substituted GRS1215 (lane 4), and E139A/E269A-substituted GRS1217 (lane 5) clones. Compressed high-molecular-weight (HMW) protein bands representing the lengthier pilus fibers along with the molecular weight markers (kDa) are identified to the left of the immunoblot.

Superposition of GG-SpaA (red) on Spy0128 (blue) (a), GBS52 (green) (b), and PitB (gold) (c). Upper structures are of superimposed N-domains. In the superposition of GG-SpaA and GBS52, the linking lysine (in sticks) and the AB loop are positioned similarly. The linking lysine and the Ω loop of Spy0128 and PitB are positioned midway along their structures. Lower structures are of full-length protein, with the C-domains superimposed and the N-domains displaced. Major structural perturbations are at the BC and DE loops in the C-domains.

(a) Schematic representation of the GG-SpaA head-to-tail arrangement in the SpaCBA pilus. Shown is the pilus-like assembly of repeating pilin molecules (orange, green, and blue) as based on the molecular packing of the GG-SpaA crystal (see ). Three pilin molecules in the asymmetric unit are highlighted. Indicated is the 120° rotation and 78 Å translation with each successive GG-SpaA pilin molecule along the pilus axis. (b) Cartoon representation of GG-SpaA pilin subunits in an assembled SpaCBA pilus structure, as deduced from the molecular packing of crystals (see ). Coloring of pilin subunits is as given above. (c) Molecular surface representation of two adjacent GG-SpaA pilin subunits in the assembled SpaCBA pilus structure. The LPXTG sorting motif of the C-terminal tail from one subunit being docked into the pilin motif-containing groove of another subunit is shown. (d) An enlarged view of the molecular interactions between two adjoining GG-SpaA pilin subunits. Upper panel shows the inter-domain interface between the C-domain one subunit (n + 1) and N-domain of the neighboring subunit (n). Lower panel shows the molecular interface between the threonine of the C-terminal LPXTG sorting motif of the n + 1 subunit and the essential lysine of the pilin motif-containing groove formed by the AB loop in the N-domain of the n subunit. Modeling of H303 and T304, which are not part of the crystal structure, brings the K171 within a distance sufficient for a covalent bond. Interface residues are depicted in ball-and-stick representation. Hydrogen bonds and salt bridges are shown as solid and dashed black lines, respectively. (e) Topology diagram showing the insertion of the C-terminal tail of one pilin into the N-domain of the adjacent pilin. The β-strands A- G are rainbow colored as in . A double-headed arrow indicates the location of an intermolecular covalent cross-link.