Important residue (G46) in erythroid spectrin tetramer formation

Abstract

0431Abbreviations used: αI-N1 -αI-spectrin fragment of residues 1-156; βI-C1 -βI-spectrin fragment of residues 1898-2083; G46A -αI-N1 variant with the G46 residue replaced by Ala; G46E -αI-N1 variant with the G46 residue replaced by Glu; G46R -αI-N1 variant with the G46 residue replaced by Arg; ITC -isothermal titration calorimetry; PBS -5 mM phosphate buffer at pH 7.4 with 150 mM sodium chloride; SpαI -erythroid α-spectrin; SpβI -erythroid β-spectrin; SpαII -non-erythroid α-spectrin; T m -temperature with 50% thermal unfolding; U mid -urea concentration with 50% unfolding Abstract: Spectrin tetramerization is important for the erythrocyte to maintain its unique shape, elasticity and deformability. We used recombinant model proteins to show the importance of one residue (G46) in the erythroid α-spectrin junction region that affects spectrin tetramer formation. The G46 residue in the erythroid spectrin N-terminal junction region is the only residue that differs from that in non-erythroid spectrin. The corresponding residue is R37. We believe that this difference may be, at least in part, responsible for the 15-fold difference in the equilibrium constants of erythroid and non-erythroid tetramer formation. In this study, we replaced the Gly residue with Ala, Arg or Glu residues in an erythroid α-spectrin model protein to give G46A, G46R or G46E, respectively. We found that their association affinities with a β-spectrin model protein were quite different from each other. G46R exhibited a 10-fold increase and G46E exhibited a 16-fold decrease, whereas G46A showed little difference, when compared with the wild type. The thermal and urea denaturation experiments showed insignificant structural change in G46R. Thus, the differences in affinity were due to differences in local, specific interactions, rather than conformational differences in these variants. An intra-helical salt bridge in G46R may stabilizeBrought to you by | MIT Libraries Authenticated Download Date | 5/10/18 2:12 PM CELLULAR & MOLECULAR BIOLOGY LETTERS 47 the partial domain single helix in α-spectrin, Helix C', to allow a more stable helical bundling in the αβ complex in spectrin tetramers. These results not only showed the importance of residue G46 in erythroid α-spectrin, but also provided insights toward the differences in association affinity between erythroid and non-erythroid spectrin to form spectrin tetramers.

Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

“…Finally, both the G46V and L49F mutations likely cause local structural perturbations as the result of substitution with comparatively bulkier residues that affect the sterics of the binding interface. Consistent with the interactions evident in the structure of the complex, the authors of previous studies have proposed that these mutations affect the binding interface (G46V) 49 or ␣-spectrin itself (L49F). 50 The locations of ␤17 mutations in the tetramer interface are shown in Figure 7.…”

Mechanism of Assembly of the Non-Covalent Spectrin Tetramerization Domain from Intrinsically Disordered Partners

“…Isothermal titration calorimetry (ITC) of the system has been performed by many groups, revealing a dissociation constant ( K d ) of about 0.4 μM for wild type. ITC has also been performed on a small subset of mutants [ 41 , 42 ]. Two studies have investigated the kinetics of tetramer formation.…”