Hydrophobic residues in small ankyrin 1 participate in binding to obscurin

Abstract

Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a ‘hot spot’ on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc_6316–6345 and Obsc_6231–6260. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc_6316–6345. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc_6231–6260. Alanine mutagenesis of the five hydrophobic residues present in Obsc_6316–6345 showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc_6231–6260 had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc_6316–6345-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc_6316–6345. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc_6316–6345 for sAnk1 and to the dominant role exhibited by this sequence in binding.

Keywords::

• Skeletal muscle
• hydrophobic interactions
• molecular dynamics

Acknowledgements

The authors acknowledge a generous grant of computer time from the Pittsburgh Supercomputing Center and National Center for Supercomputing Applications. This work was supported by stipends to CW and BB from two training grants, T32 GM08181 (RJB, PI) and T32 AR07592 (Dr M. Schneider, PI), by grants from the NIH (RO1 AR056330, to RJB; CA120215 and GM051501, to ADM) and the Muscular Dystrophy Association (to RJB), and by the University of Maryland Computer-Aided Drug Design Center.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.