Structural Characterization of a Protein A Mimetic Peptide Dendrimer Bound to Human IgG

Abstract

Understanding the chemical physical properties of protein binding sites is at the basis of the rational design of protein ligands. The hinge region of the Fc fragment of immunoglobulin G is an important and well characterized protein binding site, known to interact with several natural proteins and synthetic ligands. Here, we report structural evidence that a Staphylococcus aureus Protein A mimetic peptide dendrimer, deduced by a combinatorial approach, binds close to the Cgamma2/Cgamma3 interface of the constant fragment of a human IgG1 molecule, partially hindering the Protein A binding site. The X-ray analysis evidenced a primary binding site located between a terminal Arg residue of the ligand peptidic arm and a hydrophobic protein site consisting of Val308, Leu309, and His310. A molecular dynamic analysis of the model derived from the X-ray structure showed that in water at room temperature the complex is further stabilized by the formation of at least one more contact between a terminal Arg residue of the second arm of the peptide and the carboxylic group of a protein amino acid, such as Glu318, Asp312, or Asp280. It appears thus that stability of the Fc-dendrimer complex is determined by the synergetic formation of multiple bonds of different nature between the dendrimer arms and the protein accessible sites. The electrostatic and van der Waals energies of the complex were monitored during the MD simulations and confirmed the energetic stability of the two interactions.

Function and 3D Structure of the N-Glycans on Glycoproteins

“…The O5 and OH6 of GlcNAc in the α1-3 arm (GlcNAc-5) interact with OH4 of the Fuc residue, whereas OH4 of the α1-3 linked Man (Man-4) makes hydrogen bonds with the hydroxyl groups of the chitobiose. (vi) In the crystal structure of mouse IgG2b Fc fragment (PDB code; 2rgs, [40]), four symmetric hydrogen bonds are found between the two oligosaccharide chains. The OH3 of the α1-3 linked Man (Man-4) makes hydrogen bonds with OH2 of its counterpart β-Man (Man-3), and likewise the OH4 with OH6 (Figure 5h).…”

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities

“…Structures provide the means to see where inhibitors are binding and what they may do to the target protein structure. Combined computational and experimental X-ray crystallographic structures can efficiently and accurately identify small molecule-binding fragments important for inhibition and inform the design of new inhibitors based upon the observed ligand-bound structure (Blundell et al, 2006; Erlanson, Fesik, Hubbard, Jahnke, & Jhoti, 2016; Moiani et al, 2009; Murray & Blundell, 2010; Perry, Harris, Moiani, Olson, & Tainer, 2009; Thomas et al, 2017; Winter et al, 2012). Starting from small fragments bound to a protein, further optimization including merging adjacent fragments with a linker can lead to the generation of better inhibitors.…”

Molecular modeling to rationalize ligand–support interactions in affinity chromatography

“…In the successive years, several ligands alternative to Protein A were proposed in the literature, which relied for their development on the use of combinatorial libraries. Fassina and co-workers developed a dendrimer peptide ligand, named PAM, composed of a core of three lysine residues and four arms composed of Arg, Tyr, and Thr residues, which demonstrated to posses competitive binding properties for human and rabbit IgG with respect to Protein A [13][14][15]. A different approach was followed by Lowe and co-workers who, exploiting combinatorial libraries and molecular modeling, were able to demonstrate that relatively simple ligands, consisting of a bi-functionalized triazine molecule, could bind human IgG with energy comparable to that of Protein A [16,17].…”

“…Several recent works have focused on the study of the structure of ion-exchange resins in different conditions [26,27], while others have investigated the effect of high ligand density in hydrophobic charge induction chromatography [28] or the role of co-solvents [29] or chemically selective displacing agents [30]. In the last years, we have placed a considerable effort in the study of the interaction between affinity materials and MABs using molecular dynamics (MD) simulations [15,[31][32][33][34][35]. In particular, we have investigated the interaction between the FC fragment of human IgG, used as a molecular MAB model, and the PAM and A2P affinity ligands, both dispersed in solution and supported on agarose [32,34].…”

“…In particular, we have investigated the interaction between the FC fragment of human IgG, used as a molecular MAB model, and the PAM and A2P affinity ligands, both dispersed in solution and supported on agarose [32,34]. The modeling of the PAM ligand relied on the availability of crystallographic data [15]. The results of these studies were recently compared with experimental data with the aim of elucidating the influence of each material component, the support, the spacer, and the surface chemistry on the overall material performance of an affinity-type purification medium for the capture of IgG [33].…”