Interaction of Obscurin a with Small Ankyrin 1

Abstract

We have studied the binding of a small product of the ankyrin 1 gene (sAnk1; Ank1.5), a ∼17.5 kDa integral protein of the sarcoplasmic reticulum, to the C-terminus of obscurin A, a ∼720 kDa protein that envelopes sarcomeres at Z-disks and M-bands. Alanine scanning mutagenesis identified several lysines and arginines in the cytoplasmic sequence of sAnk1 that mediate binding to obscurin, and several glutamates in the high affinity site of obscurin that mediate binding to sAnk1. Complementary K- or R-to-E and E-to-K mutations identified specific pairs of residues involved in binding; mutagenesis of several of these eliminates binding. Molecular and Brownian dynamics simulation suggested several possible models for the docked complex but predicted only one in which D111 of sAnk1 and K6338 of obscurin interact. We confirmed their interaction by complementary mutagenesis. We tested the model further by using similar approaches to examine the hydrophobic residues involved in binding, with results consistent with the predictions of a representative structure of the docked complex selected from cluster analysis of structures generated from molecular dynamics simulations. Our studies indicate that the obscurin-binding region of sAnk1 is comprised of two ankyrin-like repeats, which establish specific electrostatic and hydrophobic contacts with the high affinity site on obscurin, composed of an 18-residue α-helical polypeptide. We identify structures similar to this polypeptide in the binding regions of proteins that interact with other ankyrin repeat proteins. We propose that the 18-mer in the high affinity binding site on obscurin for sAnk1 represents a prototypical ankyrin binding motif. Supported by grants to ADM from the NIH (CA120215; GM051501) and the University of Maryland Computer-Aided Drug Design Center, and from grants to AKK and RJB from the MDA and the NIH (RO1 AR052768 to AKK; RO1 AR056330 to RJB).