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W. Clay Smith, Daniel Turner, Elizabeth Weber, Del Benzenhafer, J. Hugh McDowell, Susan Bolch; Structural Mapping of the Interaction Between Arrestin and Enolase. Invest. Ophthalmol. Vis. Sci. 2011;52(14):50.
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We have recently reported that arrestin1 (and arrestin4) binds and modulates enolase1 in the inner segments of photoreceptors. The goal of this research is to identify the interacting domains on each protein to provide a structural understanding for this interaction and to provide an avenue for manipulating this intersection between phototransduction and glycolysis.
Domains of interaction between enolase1 and arrestin1 were mapped using three techniques: 1) arrestin pull down using heterologously expressed portions of enolase expressed from the pET-28 plasmid, 2) enolase pull down using glutathione-S-transferase (GST) fusions of portions of arrestin from pGEX-4T, and 3) tandem mass spectrometric identification of cross-linked arrestin-enolase peptides tethered by di-thiobis(succinimidylprioprionate) (DSP). In addition, conformational changes in arrestin induced by enolase binding were measured by labeling 25 site-directed cysteine mutants in loops of arrestin with monobromobimane with subsequent monitoring of fluorescence emission.
Results: Purified 145 amino acid fragments of enolase1 were blotted onto polyvinylidene fluoride and probed with full-length arrestin. Arrestin bound to residues 146-290. Using tethered enolase to bind 101 amino-acid fragments of arrestin-GST fusions resulted in selective binding of polypeptides containing arrestin residues 1-101. Tandem ms/ms of tryptic fragments of DSP cross-linked enolase/arrestin identified a single complex containing both an enolase peptide and an arrestin peptide, cross-linked at Lys-53 of arrestin and Lys-228 of enolase. Fluorimetric scanning of all arrestin loops showed an increase in fluorescence emission upon binding by enolase at loops XIV-XV, XVI-XVII, and XVII-XIX in the carboxyl domain, indicating decreased aqueous exposure of the bimane fluorophore from either burying of the bimane into arrestin or from shielding by enolase. Only in loop I-II at residue 18 did the bimane show decreased fluorescence when enolase was added, indicative of the fluorophore increasing its aqueous exposure.
Pull down assays indicate that arrestin interacts with enolase principally through the N-terminal 100 residues in arrestin and the middle third of enolase. Mass spectrometry supports this observation, showing that when arrestin is crosslinked to enolase, crosslinking occurs between Lys-53 of arrestin and Lys-228 of enolase.
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