Fig 1.
The schematic view of the studied system.
(a) Electron microscopy image of the surface of bacteria representing the length of M1 protein. The scale bar in the larger image is 500 nm where in the zoom view below is 100 nm. Small arrows indicate the position of M1 proteins. (b) The general structure of human IgG. (c) Important domains of the M1 protein, including the hypervariable domain (A), fibrinogen binding domains (B1/B2), S-region, and albumin-binding domains (C1/C2/C3). The coiled-coil region, the anchor, and the signal peptide are shown to the right specifying by the residue numbers in red. (d) IgG-orientations and -interactions with the M1-protein on the surface of GAS. Three major interactions are shown, including Fc- and Fab-mediated interactions, as well as opsonizing antibodies bound to the surface of the bacteria [16]. M1 is shown in gray while IgGs are in pink. The peptidoglycan layer on the surface of bacteria is shown in green.
Fig 2.
M1 interactions with IgG subclasses.
(a) The model for M1 (A- and S-domains)-IgG1 full length non-immune Fc-mediated interaction. Important domains of the M1 protein are shown, where A- and S-domains play a crucial role. The molecular details of the Fc-binding site are shown in panel (b) where inter- and intra-XLs are mapped on the protein complex. (b-e) The S-domain of the M1-protein interacts with the Fc-region of IgG1-4. All models are generated by the TX-MS workflow based on cross-link constraints derived from MS data. Intra-XLs for each IgG are shown in cyan, while inter-XLs between the two proteins are in blue. The binding interfaces of the M1 protein on the Fc-domain of all IgG subclasses are similar and mediated via the CH3 domains. This region is involved in binding to IgG Fc-receptors (FcγR), predicted to be inhibited by the M1 protein interaction.
Fig 3.
The TX-MS based M1-IgG model aligned with that of the streptococcal protein G.
(a-b) Protein G (PDB ID: 1FCC) is shown in red with its binding interface on IgG1 in dark blue. The M1 protein is shown in magenta and dark grey with its binding interface on IgG in light blue. Both proteins share the same binding site on IgGs in CH3 domains and close to CH2.
Fig 4.
DDA-based peptide quantification of Fc and Fab fragments of IgG subclasses bind to different sub-domains of M1 protein.
All IgG subclasses bind to the M1 protein through their Fc-domains. The S- & C1-domains and the hypervariable domain A of the M1 protein have the highest affinity for IgG subclasses. Only IgG heavy chains were considered.
Table 1.
Peptides with high-affinity binding occurrences.
The listed peptides are frequent in the binding interfaces of the M1 protein and different IgGs according to the XL-MS quantification.
Fig 5.
H-bond network between the M1 peptides and IgG Fc-domain.
The residues forming H-bond contacts between the (a) M1(A)-IgG(Fc) and (b) M1(S)-IgG(Fc) are shown with spheres. The thickness of the yellow lines corresponds to the persistence of the interactions throughout the replicates of MD simulations. The M1 peptide is colored in gray while the chains of the IgG Fc-domain are depicted in red and pink. The distribution of distances between the residues forming H-bond contacts along the MD simulations are shown for the (c) M1(A)-IgG(Fc) and (d) M1(S)-IgG(Fc). The number of replicates in which, the H-bonds are formed for at least 40% of the simulation time are added in red at the top of each box. Communication Blocks (CBspath) identified by COMMA2 are mapped on the structure of (e) M1(A)-IgG(Fc), and (f) M1(S)-IgG(Fc). Distinct CBspath of four residues or more are highlighted in different colors. The isolated communication pathway between the M1 peptide A-domain (T110) and IgG Fc-domain (E263) is depicted by a black line on the corresponding structure.