Ankyrin binding motifs: Lessons from sAnk1-obscurin binding

My research examines the molecular details of the binding of obscurin to small ankyrin 1 (sAnk1). sAnk1 is an integral membrane protein of the sarcoplasmic reticulum, the cytosolic region of which is believed to contain ankyrin repeats, though they are not predicted by computational algorithms. Obscurin A is a large modular protein containing 6620 amino acids that binds sAnk1. Two binding sites for sAnk1 have been identified on obscurin, the first between residues 6235 and 6260 and the second between 6316 and 6436. I have compared and contrasted the binding to sAnk1 of oligopeptides and small fusion proteins that include these sequences and constructed preliminary structural models for both of these sites. I determined that residues 6316-6345 of obscurin have a higher affinity for the cytosolic region of sAnk1 than residues 6231-6260. I also used site-directed mutagenesis to identify the specific charged residues in the sequence between amino acids 6316 and 6436 that are necessary for binding, assaying binding by blot overlays and surface plasmon resonance. Using a similar approach in dual cycle mutagenic studies, I identified the complementary residues of opposite charge on sAnk1 with which they interact. Studies utilizing a molecular dynamics approach determined the most likely structures of sAnk1 and its high-affinity binding region of obscurin and refined the structure of the docked complex, selected from a larger number of possible structures by its agreement with experimental data. Molecular dynamics also confirmed the partial alpha-helical character of obscurin 6316-6345, which I determined initially with circular dichroism studies and computational modeling. I used similar methods to study the sequence of the lower affinity binding site on obscurin, between residues 6231 and 6260. I then identified short sequences in several other human proteins that are known to bind ankyrin repeat proteins. I show that these comprise at least distinct 3 types of ankyrin binding motifs and present preliminary experiments to support my predictions for the two types found in the retinoblastoma protein, Rb, and an examples of one type found in histone deacetylase 4, that resemble the motifs I have studied in obscurin. My studies show that the binding of ankyrin binding motifs to proteins with ankyrin repeats is mediated by a combination of highly specific electrostatic, and probably hydrophobic, interactions. They further suggest that the methods I have developed to identify distinct classes of ankyrin binding motifs can be used to computationally identify many other small protein domains that mediate interaction with other common protein folds.