title: The beta4-beta8 groove is an ATP-interactive site in the alpha crystallin core domain of the small heat shock protein, human alphaB crystallin.
abstract: The site for ATP interactions in human alphaB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the alphaB crystallin sequence, (82)KHFSPEELKVKVLGD(96) as a Walker-B ATP-binding motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[gamma]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human alphaB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the beta4-beta8 groove as the ATP interactive site in alphaB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the beta8 strand ((131)LTITSSLS(138)). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the alphaB crystallin Walker-B motif photo-affinity-labeled with azido-ATP-EDANS confirming the beta4-beta8 groove as an ATP interactive site. The results presented here clearly establish the beta4-beta8 groove as the ATP interactive region in alphaB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.
authors.affiliation: Biomolecular Structure and Design, University of Washington, Seattle, WA 98195-7420, USA.
authors.forename: Joy G
authors.initials: JG
authors.lastname: Ghosh
authors.forename: Scott A
authors.initials: SA
authors.lastname: Houck
authors.forename: Catalin E
authors.initials: CE
authors.lastname: Doneanu
authors.forename: John I
authors.initials: JI
authors.lastname: Clark
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics
chemical_list: D038203:alpha-Crystallin B Chain; D000255:Adenosine Triphosphate