The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin

The site for ATP interactions in human αB 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 αB crystallin sequence, 82KHFSPEELKVKVLGD96 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-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB 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 β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.