The beta4-beta8 groove is an ATP-interactive site in the alpha crystallin core domain of the small heat shock protein, human alphaB crystallin.
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.
Ghosh
Joy G
JG
Biomolecular Structure and Design, University of Washington, Seattle, WA 98195-7420, USA.
Houck
Scott A
SA
Doneanu
Catalin E
CE
Clark
John I
JI
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
D038203:alpha-Crystallin B Chain; D000255:Adenosine Triphosphate