title: Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
abstract: Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit-subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker-containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d₀ and d₄) cross-linker-BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88-K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Rama
authors.initials: R
authors.lastname: Kannan
authors.forename: Puttur
authors.initials: P
authors.lastname: Santhoshkumar
authors.forename: Brian P
authors.initials: BP
authors.lastname: Mooney
authors.forename: K Krishna
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry; D005977:Glutarates / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D013388:Succinimides / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003432:Cross-Linking Reagents; D005977:Glutarates; D050505:Mutant Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D013388:Succinimides; D038202:alpha-Crystallin A Chain; C427145:bis(sulfosuccinimidyl)glutarate