Mapping the binding site on small ankyrin 1 for obscurin.
Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.
Borzok
Maegen A
MA
Department of Biochemistry and Molecular Biology, University of Maryland, School of Medicine, Baltimore 21201, USA.
Catino
Dawn H
DH
Nicholson
James D
JD
Kontrogianni-Konstantopoulos
Aikaterini
A
Bloch
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D017385:Sequence Homology; D020798:Two-Hybrid System Techniques
C110202:ANK1 protein, human; D017487:Ankyrins; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C578224:OBSCN protein, human; D017346:Protein Serine-Threonine Kinases