Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology

Abstract

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.

Streptococcus pyogenes Forms Serotype- and Local Environment-Dependent Interspecies Protein Complexes

“…The site engaged in binding will block the deuterium exchange, which is then used to map the binding interface. As the input, we first generated a computational model of full-length M28, which was determined using the Rosetta comparative modeling (RosettaCM) protocol ( 45 ), based on the previously reported model of the M1 protein ( 27 ). This model was further used to provide protein-protein docking decoys using structures deposited in the Protein Data Bank for IgA (PDB accession number 6LXW ) and C4BP (PDB accession number 5HYP ).…”

“…The UniProt accession numbers used for the S. pyogenes M28 protein and human C4BPa, C4BPb, IGHA1, and IGHA2 are W0T1Y4 , P04003 , P20851 , P01876 , and P01877 , respectively. The tertiary structure of the M28 protein was characterized using the Rosetta comparative modeling (RosettaCM) protocol ( 45 ) from the Rosetta software suite ( 68 ) based on the previously generated full-length model of the M1 protein ( 27 ) as the homologue structure. For IgA and C4BP, PDB accession numbers 6LXW and 5HYP , respectively, were used.…”

“…The orientation of IgG binding, i.e., whether it is Fab or Fc mediated, is governed by the concentration of IgGs in the host niche ( 16 , 26 ). Binding to IgG Fc is mediated by the S region found in some M proteins and located between the B and C repeats and the HVR ( 15 , 27 ). Other M proteins such as M4 and M22 of emm pattern E have been shown to bind immunoglobulin A (IgA) ( 16 , 28 ).…”

Proteomic Approaches to Unravel Mechanisms of Antibiotic Resistance and Immune Evasion of Bacterial Pathogens

“…Khakzad et al developed an affinity procedure preceded by a chemical cross-linking on human blood plasma using live S. pyogenes to characterize the multicomponent human complement system membrane attack complex (MAC) associated with the bacterial surface and provided detailed information of protein–protein complexes in their native environment ( 103 ). In the same line, the same authors, early this year combined a targeted XL-MS approach with molecular dynamics (MD) simulations to characterize the S. pyogenes M1 protein (virulence factor) and human-IgG interactions ( 102 ). Accordingly, XL-MS revealed binding interfaces, while the MD simulations helped to elucidate the interaction network in molecular detail.…”

“…The same Malmström group determined the Fc-binding interface, demonstrating a specific site in the IgG CH3 domain (essential for binding to FcγR receptor). Mimicking an ex vivo scenario during invasive infections, these interactions revealed binding with the non-immune Fc-domain, locking the FcγR receptor interaction, and assisting the bacteria in evasion from phagocytic killing ( 102 ).…”