Characterization and Comparison of Two Binding Sites on Obscurin for Small Ankyrin 1

Obscurin A, an ∼720 kDa modular protein of striated muscles, binds to small ankyrin 1 (sAnk1, Ank 1.5), an integral protein of the sarcoplasmic reticulum, through two distinct carboxy-terminal sequences, Obsc6316−6436 and Obsc6236−6260. We hypothesized that these sequences differ in affinity but that they compete for the same binding site on sAnk1. We show that the sequence within Obsc6316−6436 that binds to sAnk1 is limited to residues 6316−6345. Comparison of Obsc6231−6260 to Obsc6316−6345 reveals that Obsc6316−6345 binds sAnk1 with an affinity (133 ± 43 nM) comparable to that of the Obsc6316−6436 fusion protein, whereas Obsc6231−6260 binds with lower affinity (384 ± 53 nM). Oligopeptides of each sequence compete for binding with both sites at half-maximal inhibitory concentrations consistent with the affinities measured directly. Five of six site-directed mutants of sAnk1 showed similar reductions in binding to each binding site on obscurin, suggesting that they dock to many of the same residues of sAnk1. Circular dichroism (CD) analysis of the synthetic oligopeptides revealed a 2-fold greater α-helical content in Obsc6316−6346, ∼35%, than Obsc6231−6260, ∼17%. Using these data, structural prediction algorithms, and homology modeling, we predict that Obsc6316−6345 contains a bent α-helix of 12 amino acids, flanked by short disordered regions, and that Obsc6231−6260 has a short, N-terminal α-helix of 4−5 residues followed by a long disordered region. Our results are consistent with a model in which both sequences of obscurin differ significantly in structure but bind to the ankyrin-like repeat motifs of sAnk1 in a similar though not identical manner.