Figure 3.
Domain structure of the N-terminal region of bovine α-spectrin with E91K substitution. (A) Domain structures of bovine spectrin recombinants used in this study, the N-terminal repeats, α[0-1] (α[0-1]), and mini-spectrin, α[0-5]–β[16-17] (mini-Sp), prepared according to bovine α[0-1] demonstrated here represents that from the allele SpαB or SpαBK91 with whose difference in the 91st residue, E91 derived from SpαB and K91 from SpαBK91. The sequence is found in alignment with that of the human RBC α[0-1], and the identical amino acid residues are indicated by asterisks. The 91st residue (E91 or K91), 2 tryptophan residues (W59 and W131), and amino acid residues that differ between SpαA and SpαB alleles (E17/A17, H87/N87, and W139/Q139) are also indicated. Prediction of the secondary structure for bovine α[0-1] by the Jpred4 software (http://www.compbio.dundee.ac.uk/jpred4) revealed its similarity to the structure that was determined by the solution NMR (nuclear magnetic resonance) structural study for human α[0-1] (unbound state, PDB ID, 1OWA)36 and by crystal structural study for α[0-1]–β[16-17] complex (bound state, PDB ID, 3LBX).22 The α-helix C′ in α0 and helices A, B, and C in α1 according to 1OWA are boxed in black rectangles, whereas the α-helices predicted for α[0-1]–β[16-17] complex according to 3LBX are found in red rectangles. (B-C) Three-dimensional structures of bovine α[0-1]E91 (illustrated in cyan) and α[0-1]K91 (illustrated in salmon) predicted by the Phyre2 software35 using 1OWA in panel B or 3LBX in panel C as templates. α-Helices C′ in α0, and A, B, and C in α1 are indicated. The 91st amino acid residue E91 (blue) or K91 (red) and conserved specific side chains that make contact with β-spectrin at the interface of the dimer-dimer self-association22 are illustrated with spheres and sticks, respectively. (D) Interhelical hydrogen bonds are depicted by the PyMOL software for the bound states of α[0-1]E91 (left) and α[0-1]K91 (right). Note that α[0-1]E91 contains interhelical hydrogen bonds involving E91–K63 and S96–W131 in the vicinity of the E91K substitution site (illustrated in magenta dotted lines), whereas these contacts are lost in α[0-1]K91. PDB ID, Protein Data Bank identifier.

Domain structure of the N-terminal region of bovine α-spectrin with E91K substitution. (A) Domain structures of bovine spectrin recombinants used in this study, the N-terminal repeats, α[0-1] (α[0-1]), and mini-spectrin, α[0-5]–β[16-17] (mini-Sp), prepared according to bovine α[0-1] demonstrated here represents that from the allele SpαB or SpαBK91 with whose difference in the 91st residue, E91 derived from SpαB and K91 from SpαBK91. The sequence is found in alignment with that of the human RBC α[0-1], and the identical amino acid residues are indicated by asterisks. The 91st residue (E91 or K91), 2 tryptophan residues (W59 and W131), and amino acid residues that differ between SpαA and SpαB alleles (E17/A17, H87/N87, and W139/Q139) are also indicated. Prediction of the secondary structure for bovine α[0-1] by the Jpred4 software (http://www.compbio.dundee.ac.uk/jpred4) revealed its similarity to the structure that was determined by the solution NMR (nuclear magnetic resonance) structural study for human α[0-1] (unbound state, PDB ID, 1OWA)36 and by crystal structural study for α[0-1]–β[16-17] complex (bound state, PDB ID, 3LBX).22 The α-helix C′ in α0 and helices A, B, and C in α1 according to 1OWA are boxed in black rectangles, whereas the α-helices predicted for α[0-1]–β[16-17] complex according to 3LBX are found in red rectangles. (B-C) Three-dimensional structures of bovine α[0-1]E91 (illustrated in cyan) and α[0-1]K91 (illustrated in salmon) predicted by the Phyre2 software35 using 1OWA in panel B or 3LBX in panel C as templates. α-Helices C′ in α0, and A, B, and C in α1 are indicated. The 91st amino acid residue E91 (blue) or K91 (red) and conserved specific side chains that make contact with β-spectrin at the interface of the dimer-dimer self-association22 are illustrated with spheres and sticks, respectively. (D) Interhelical hydrogen bonds are depicted by the PyMOL software for the bound states of α[0-1]E91 (left) and α[0-1]K91 (right). Note that α[0-1]E91 contains interhelical hydrogen bonds involving E91–K63 and S96–W131 in the vicinity of the E91K substitution site (illustrated in magenta dotted lines), whereas these contacts are lost in α[0-1]K91. PDB ID, Protein Data Bank identifier.

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