Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes

Abstract

The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2-3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2-3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2-3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.

Keywords: Adhesin; Antigen I/II; Isopeptide bond; X-ray crystallography.

Fig. 1

Domain organization of S. pyogenes AspA and sequence alignment of the C_2–3 domains of AspA, SpaP and SspB. (A) The primary sequence of AspA begins with a signal peptide (SP), followed in sequence by a small N-terminal domain (N), an alanine rich repeat region (A), a variable domain (V), a proline rich repeat region (P), a C-terminal region (C), and finally a cell wall anchor segment (CW) containing an LPxTG-motif for sortase mediated cell wall attachment. (B) Sequence alignment of the AspA (Uniprot id. Q48S75), SpaP (P23404) and SspB (P16952) C_2–3 domains. Secondary structure elements from the structure of AspA-C_2–3 are marked above the alignment.

Fig. 2

Overall topology of AspA-C_2–3. (A) Ribbon diagram of AspA-C_2–3 presented in stereo. The C₂ domain (residues 971–1150) is depicted in blue while the C₃ domain (1151–1306) is depicted in yellow. A bound calcium ion is shown as a grey sphere. (B) Topology diagram of AspA-C_2–3 where α-helices are represented as rectangles, β-strands as arrows and loops as lines. The isopeptide bonds between K987 and N1128 and between K1155 and N1286 are marked as red lines. (C) Electrostatic surface potential rendering of AspA-C_2–3 colored from −0.5 V (red) to 0.5 V (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Isopeptide bonds and metal binding site. (A) The isopeptide bond between K978 on strand A and N1128 on strand F in the C₂ domain is represented as a stick model in a 2Fo-Fc map, contoured at 0.90 e/ų. It is stabilized by hydrogen bonding with the side chain of D1028 (dashed lines). (B) In the C₂ domain a metal ion, modeled as a Ca²⁺ ion, is coordinated by D1029 and Y1030 on the strands C-D loop, V1084 and E1086 from the loop connecting helix DH1 with strand D″, and one water molecule. The figure is presented in stereo.

Fig. 4

Comparative structural and evolutionary conservation analyses of AspA-C_2–3. (A) Superposition of the AspA-C_2–3 structure (blue) with the structures of SspB-C_2-3 from S. gordonii (orange, PDB: 2WOY) and SpaP-C_2–3 from S. mutans (green, PDB: 3OPU). The BAR (SspB Adherence Region), recognized by P. gingivalis short fimbrial protein Mfa1, is highlighted by a black circle and is shown in more detail as a stereo image in (B) together with a sequence alignment of the BAR helix. (C) Space filling model representation of evolutionary conservation analyses for AspA-C_2–3 performed using ConSurf . The level of conservation of individual amino acids is indicated from variable (turquoise, 1) to highly conserved (maroon, 9) according to the color coding bar. Positions for which the level of conservation was assigned with low confidence are marked with yellow color. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)