Energetic and dynamic characterization of the IgA1:FcαRI interaction reveals long-range conformational changes in IgA1 upon receptor binding

The interaction between immunoglobulin A (IgA) and the IgA-specific receptor, FcαRI, initiates a pro-inflammatory immune response in the presence of a multivalent antigen, and has recently been shown to possess an anti-inflammatory role in the absence of antigen. Additionally, soluble FcαRI has been implicated in the disease progression of IgA nephropathy (IgAN), the most common form of glomerulonephritis, characterized by an aberrant IgA1 glycosylation profile and the deposition of IgA1-containing complexes in the kidney, resulting in end-stage renal failure in 20-30% of IgAN patients. The mechanism of FcαRI's involvement in IgAN, however, is unclear. Naturally-occurring circulating immune complexes consisting of IgA-bound FcαRI have been implicated in IgAN, although soluble FcαRI levels are similar to those of healthy individuals. The studies described herein were focused on elucidating the energetic and dynamic elements inherent to the FcαRI:IgA interaction, with the ultimate goal of understanding the interaction at the molecular level. This knowledge will provide an important basis for the potential development of future therapeutics targeted at inflammatory responses, as well as IgAN.Utilizing the crystal structure of the 2:1 complex between FcαRI and IgA1-Fc (Fcα), we identified 10 IgA1 residues with side-chain contacts with FcαRI. FcαRI binds IgA at the Cα2-Cα3 junction; the majority of IgA side-chain contacts are contributed by the Cα3 domain, except for two from the Cα2 domain. These residues were individually mutated to alanine and we systematically characterized the energetic contributions of each residue to this interaction using biosensor analyses. We have identified key energetic residues to be centrally-positioned hydrophobic contacts, as well as Cα2 domain residues, located at the periphery of the interface. Each residue was necessary for stable complex formation. Comparison of our biosensor results to other Ig:FcR interactions revealed overlapping binding footprints and a conserved mode of binding.Dynamic characterization of the FcαRI:Fcα interaction revealed that binding of a single FcαRI molecule to Fcα dampens dynamic motions of both the bound and the unbound Fcα chains, suggesting that conformational changes are communicated across the entire Fcα molecule. This is consistent with the apparent negative cooperativity we observed in our biosensor analyses with immobilized Fcα, where the second FcαRI binding event exhibited a weaker affinity than the first binding event. To experimentally validate the computational analyses and investigate the potential for long-range conformational changes to affect binding elsewhere on the IgA1 molecule, we conducted biosensor experiments using full-length IgA1. We measured binding of Helix aspersa agglutinin (HAA), a lectin that specifically recognizes terminal GalNAc moieties on IgA1 hinge O-glycans, to the IgA1 hinge in the presence and absence of FcαRI. FcαRI binding to the Fc domain of IgA1 increased the affinity of HAA for the IgA1 hinge, conclusively showing that long-range conformational changes are communicated from Fc domain to the hinge. Taken together, these studies suggest that, in IgAN, circulating FcαRI:IgA1 complexes may expose the IgA1 hinge, preferentially presenting hinge epitopes to naturally-occurring IgA/IgG autoantibodies. This would facilitate the formation of large IgA-containing complexes that can deposit in the kidney and subsequently contribute to renal damage.

Name thesis:degree_name

PhD

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Medicine: Molecular Genetics, Biochemistry, and Microbiology

Grantor dc:publisher

University of Cincinnati

Year dc:date

2012

Author dc:creator

• Posgai, Monica Therese

Contributors dc:contributor

• Herr, Andrew

• Biology
• Biophysics
• Immunology
• Molecular Biology
• Molecules
• Fca
• RI, Immunoglobulin A, surface plasmon resonance, energetics, dynamic, normal mode analysis

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=ucin1354043317

OAI identifier oai:identifier

oai:etd.ohiolink.edu:ucin1354043317