title: Hydrophobic residues in small ankyrin 1 participate in binding to obscurin.
abstract: 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, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ 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 Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. 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 Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.
authors.affiliation: Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD, USA.
authors.forename: Chris D
authors.initials: CD
authors.lastname: Willis
authors.forename: Taiji
authors.initials: T
authors.lastname: Oashi
authors.forename: Ben
authors.initials: B
authors.lastname: Busby
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Mackerell
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bloch
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: C540195:Ank1 protein, rat; D017487:Ankyrins; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; C503780:Obscn protein, rat; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007930:Leucine; D000409:Alanine