title: Identification and characterization of a copper-binding site in αA-crystallin.
abstract: Previous studies have shown that both αA- and αB-crystallins bind Cu2+, suppress the formation of Cu2+-mediated active oxygen species, and protect ascorbic acid from oxidation by Cu2+. αA- and αB-crystallins are small heat shock proteins with molecular chaperone activity. In this study we show that the mini-αA-crystallin, a peptide consisting of residues 71-88 of αA-crystallin, prevents copper-induced oxidation of ascorbic acid. Evaluation of binding of copper to mini-αA-crystallin showed that each molecule of mini-αA-crystallin binds one copper molecule. Isothermal titration calorimetry and nanospray mass spectrometry revealed dissociation constants of 10.72 and 9.9 μM, respectively. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid interaction with mini-αA-crystallin was reduced after binding of Cu2+, suggesting that the same amino acids interact with these two ligands. Circular dichroism spectrometry showed that copper binding to mini-αA-crystallin peptide affects its secondary structure. Substitution of the His residue in mini-αA-crystallin with Ala abolished the redox-suppression activity of the peptide. During the Cu2+-induced ascorbic acid oxidation assay, a deletion mutant, αAΔ70-77, showed about 75% loss of ascorbic acid protection compared to the wild-type αA-crystallin. This difference indicates that the 70-77 region is the primary Cu2+-binding site(s) in human native full-size αA-crystallin. The role of the chaperone site in Cu2+ binding in native αA-crystallin was confirmed by the significant loss of chaperone activity by the peptide after Cu2+ binding.
authors.affiliation: Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
authors.forename: Murugesan
authors.initials: M
authors.lastname: Raju
authors.forename: Puttur
authors.initials: P
authors.lastname: Santhoshkumar
authors.forename: T Michael
authors.initials: TM
authors.lastname: Henzl
authors.forename: K Krishna
authors.initials: KK
authors.lastname: Sharma
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D038202:alpha-Crystallin A Chain; D003300:Copper; D001205:Ascorbic Acid