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Blood, Vol. 113, Issue 22, 5377-5384, May 28, 2009
The structure of the ankyrin-binding site of β-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties
Blood Stabach et al.
113: 5377
Supplemental materials for: Stabach et al
Files in this Data Supplement:
- Table S1. Oligonucleotide primers used to generate mutant βI-14.15 peptides (PDF, 4.24 MB)
- Table S2. Relative ankyrin binding of spectrin βI-14,15 mutations (PDF, 725 KB)
- Table S3. Data collection, phasing, and refinement statistics (PDF, 529 KB)
- Figure S1. Comparison of sequence divergence within βI spectrin and across other β- spectrins (JPG, 219 KB)
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(A) Alignment of βI-spectrin repeats within βI spectrin. (B) Comparison of linker sequences across all β-spectrins.
- Figure S2. Comparison of canonical triple helical sequences of human spectrins identifies the sites of frequent sequence divergence or insertion (JPG, 1.22 MB)
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Each spectrin repeat consists of an A, B, and C helix. Each helix is composed of multiple heptad repeats. When position “a” or “d” of the heptad repeat is non-polar the residue is highlighted in cyan. The only exception is the arginine residue in the “d” position of the 4th heptad of the C helix. Because the long side chains of charged amino acids have been shown to satisfy the requirements of a non-polar residue, this Arg is considered non-polar and colored cyan. A basic amino acid kink (yellow) disrupts the periodicity of the B helix. Helices A and B are connected by an A–B loop and helices B and C are connected by a B–C loop. An alpha helical linker connects the end of one C helix with the beginning of the next A helix. Loops and linkers are colored red. Insertions or deletions that disrupt the register of adjacent repeats are underlined and occur mainly in 4 places; the A–B loop, the basic amino acid kink, the B–C Loop, and in the linker. These comparisons highlight how evolutionary diversity is concentrated in the C-helix-linker and B–C loops of the spectrin repeats. These regions however are not devoid of pressure to be conserved, since they tend to be well conserved across different spectrins (c.f. Fig. S1). The alpha spectrins (A, B): Domain I of α-spectrin is a short stretch of amino acids before the beginning of helical structure that can be considered a partial repeat consisting of a C helix only. Domain II contains 20 full repeats. Both α-spectrins contain an 8 residue insertion in the linker between repeats 18 and 19 and again between repeats 19 and 20. Domain III contains two EF Hand regions (A) Alpha I-spectrin Repeat 9 contains an SH3 domain inserted in the B–C loop and another insertion in the B–C loop of repeat 17. (B) Alpha II-spectrin Repeat 9 contains an SH3 domain inserted in the B/C Loop and another large insert in the middle of the C helix. Repeat 10 contains an insert in the middle of the C helix that harbors the micro calpain cleavage site and a calmodulin-binding domain. Repeat 14 contains an insertion in both the A/B Loop and the B/C Loop. Repeat 20 contains an insertion in the A/B Loop Beta Spectrins (C–F): Domain I of the Beta I, II, III, IV spectrins contains an actin binding domain with two calponin homology domains in BLUE. Domain II contains 16 full repeats and a partial repeat 17 consisting of only an A and a B helix. All of these beta spectrins contain an 8 residue insertion in the linker between repeats 1 and 2. Domain III contains a pleckstrin homology (PH) domain highlighted in Blue (C) Beta I-spectrin contains an insert in the B–C loop of repeat 1, 3, 8, and 15 as well as a single amino acid insertion in the A–B loop of repeat 7. Repeat 11 has a 7 amino acid deletion that falls near the B–C loop. (D) Beta II-spectrin contains an insert in the B–C loop of repeats 1, 3, 8, 12, and 15 as well as a single amino acid insertion in the A–B loop of repeat 7. (E) Beta III-spectrin contains an insert in the B–C loop of repeats 1, 3, 8, and 15 as well as a single amino acid insertion in the A–B loop of repeat 7. Repeat 12 has a 6 amino acid deletion that falls in the B–C loop. (F) Beta IV Spectrin contains an insert in the B–C loop of repeats 1, 4, 5, 8, and 15 as well as an insertion in the A–B loop of repeats 4 and 8.
- Video 1. Structure of the βI-14,15 two-repeat unit rotating about its long-axis (MOV, 4.37 MB)
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The molecular surface and the coil representation of the βI spectrin di-repeat structure are shown. Coloration is as in Fig. 3. Solvent-exposed residues critical for ankyrin recognition are red; buried residues are green; the remaining residues are colored light blue.
- Video 2. Hypothesized two-state transition in the ankyrin-binding domain (MOV, 1.36 MB)
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The mutagenesis and structural data indicate that the acute angle between the two subunits in βI-14,15 is required for effective ankyrin binding. It is proposed that mechanical stress and/or ligand binding that modifies the relationship of the two subunits is a potentially general mechanism by which spectrin di-repeat units can transduce mechanical stress or deformation into alterations in protein-ligand binding and vice-versa. Such a mechanism may also propagate an allosteric signal along the spectrin scaffold, a process possibly relevant to the coupling between ankyrin-binding and spectrin self-association. The trajectory of the conformational change was generated in Lsqman, the frames were made in Pymol and the movie was produced in QuickTimePro. The coloring scheme is the same as in Fig. 6, with the putative ankyrin-binding surface colored red.
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