title: Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor.
abstract: Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.
authors.affiliation: From the Departments of Protein Analytical Chemistry,; Early Stage Pharmaceutical Development, and. Electronic address: Walters.Benjamin@gene.com.
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Walters
authors.affiliation: the Department of Pharmacy, University of Copenhagen, 1165 Copenhagen, Denmark.
authors.forename: Pernille F
authors.initials: PF
authors.lastname: Jensen
authors.affiliation: Roche Pharma Research and Early Development (pRED), Roche Innovation Center, DE-82377 Penzberg, Germany, and.
authors.forename: Vincent
authors.initials: V
authors.lastname: Larraillet
authors.affiliation: Analytical Operations, Genentech Inc., South San Francisco, California 94080-4990.
authors.forename: Kevin
authors.initials: K
authors.lastname: Lin
authors.affiliation: Early Stage Pharmaceutical Development, and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Patapoff
authors.affiliation: Roche Pharma Research and Early Development (pRED), Roche Innovation Center, DE-82377 Penzberg, Germany, and.
authors.forename: Tilman
authors.initials: T
authors.lastname: Schlothauer
authors.affiliation: the Department of Pharmacy, University of Copenhagen, 1165 Copenhagen, Denmark.
authors.forename: Kasper D
authors.initials: KD
authors.lastname: Rand
authors.affiliation: From the Departments of Protein Analytical Chemistry.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Zhang
mesh_terms: D002846:Chromatography, Affinity; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; C100688:Fc receptor, neonatal
keywords: HDX-MS; affinity; antibody; biophysics; conformational dynamics; hydrogen exchange mass spectrometry; immunoglobulin G (IgG); neonatal Fc receptor (FcRn); protein stability; structural biology