Binding characteristics of staphylococcal protein A and streptococcal protein G for fragment crystallizable portion of human immunoglobulin G

Abstract

In the wide array of physiological processes, protein-protein interactions and their binding are the most basal activities for achieving adequate biological metabolism. Among the studies on binding proteins, the examination of interactions between immunoglobulin G (IgG) and natural immunoglobulin-binding ligands, such as staphylococcal protein A (spA) and streptococcal protein G (spG), is essential in the development of pharmaceutical science, biotechnology, and affinity chromatography. The widespread utilization of IgG-spA/spG binding characteristics has allowed researchers to investigate these molecular interactions. However, the detailed binding strength of each ligand and the corresponding binding mechanisms have yet to be fully investigated. In this study, the authors analyzed the binding strengths of IgG-spA and IgG-spG complexes and identified the mechanisms enabling these bindings using molecular dynamics simulation, steered molecular dynamics, and advanced Poisson-Boltzmann Solver simulations. Based on the presented data, the binding strength of the spA ligand was found to significantly exceed that of the spG ligand. To find out which non-covalent interactions or amino acid sites have a dominant role in the tight binding of these ligands, further detailed analyses of electrostatic interactions, hydrophobic bonding, and binding free energies have been performed. In investigating their binding affinity, a relatively independent and different unbinding mechanism was found in each ligand. These distinctly different mechanisms were observed to be highly correlated to the protein secondary and tertiary structures of spA and spG ligands, as explicated from the perspective of hydrogen bonding.

Keywords: AFM, Atomic Force Microscopy; APBS, Advanced Poisson–Boltzmann Solver; Affinity chromatography; BIR, Between Protein–Protein Interface Residues; ELISA, Enzyme-linked Immunosorbent Assays; Fc, Fragment Crystallizable; IgG, Immunoglobulin G; Immunoglobulin G; MD, Molecular Dynamics; MM/PBSA, Molecular Mechanics Poisson–Boltzmann Surface Area; Molecular dynamics; Protein A; Protein G; Protein docking; RMSD, Root Mean Square Deviation; SASA, Solvent Accessible Surface Area; SMD, Steered Molecular Dynamics; spA, Staphylococcal Protein A; spG, Streptococcal Protein G.

Graphical abstract

Fig. 1

Three-dimensional representation of protein crystalline structure between IgG Fc portion and proteins (a) A or (b) G; orange and yellow ribbons indicate spA and spG ligands, respectively. Two heavy chains in IgG Fc portion are marked with green and cyan ribbons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

RMSD of complexes calculated during 50 ns of MD simulations from initial conformations for (a) IgG–spA and (b) IgG–spG complexes; RMSD values of protein–protein interface residues and each heavy chain of IgG Fc domain are plotted in (c) and (d) with schematic of configuration pose of each complex on right.

Fig. 3

Hydrogen bond contribution to stable crystalline binding structures: (Left) Highlighted key pair of hydrogen bonding residues; pink dotted line in middle inserts indicates key hydrogen bonds; (Right) number of hydrogen bonds formed between protein–protein interface residues (BIR) and non-BIR for (a) IgG–spA or (b) IgG–spG complex over time (BIR ratio obtained by dividing BIR hydrogen bond by total number of hydrogen bonds). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Calculated forces over time to undock (a) spA ligand or (b) spG ligand from IgG binding site, and number of hydrogen bonds formed between inter-proteins and intra-protein for corresponding time. Inter-protein ratio is obtained by dividing the inter-protein hydrogen bond by total number of hydrogen bonds. Unbinding pathway consists of four sequence processes. For each process, representative conformations of each complex are shown in figure inserted above.

Fig. 5

Electrostatic potential maps of (a) IgG–spA and (b) IgG–spG complexes where interaction site is expressed on horizontal plane. Blue area has high potential with relatively no electrons (positive charge). Red area has low potential and relatively abundant electrons (negative charge). White area between red and blue has neutral charge. Right rotational inserts show electrostatic interaction of complexes on each ligand or receptor binding pocket. Green dotted line on electrostatic surface show location of protein–protein interface residues. Detailed configuration poses of each protein are represented by transparent surface maps. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Mapping hydrophobicity of each protein surface. Surface hydrophobicity map of protein–protein interface residues is opaque and surrounded by green dotted line. Orange and blue portions denote the most hydrophobic and hydrophilic areas, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Binding free energy of protein–protein interface residues in (a) spA and (b) spG ligands. Gray arrows indicate protein secondary segment to which residue belongs. Helix 1α or 1β structure is segment of interest in which peculiar unbinding conformation is observed.