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.

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38-284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697-2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the Cterminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the K d values for V22F were similar to those for the wild-type (about 7 nM), whereas the K d values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of non-erythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.