- Short Communication
- Open Access
Important residue (G46) in erythroid spectrin tetramer formation
Cellular & Molecular Biology Letters volume 15, Article number: 46 (2009)
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 stabilize 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.
αI-spectrin fragment of residues 1–156
βI-spectrin fragment of residues 1898–2083
αI-N1 variant with the G46 residue replaced by Ala
αI-N1 variant with the G46 residue replaced by Glu
αI-N1 variant with the G46 residue replaced by Arg
isothermal titration calorimetry
5 mM phosphate buffer at pH 7.4 with 150 mM sodium chloride
- Tm :
temperature with 50% thermal unfolding
- Umid :
urea concentration with 50% unfolding
Bennett, V. and Healy, J. Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin. Trends Mol. Med. 14 (2008) 28–36.
Mohandas, N. and An, X. New insights into function of red cell membrane proteins and their interaction with spectrin-based membrane skeleton. Transfus. Clin. Biol. 13 (2006) 29–30.
Elgsaeter, A., Stokke, B.T., Mikkelsen, A. and Branton, D. The molecular basis of erythrocyte shape. Science 234 (1986) 1217–1223.
Begg, G.E., S.L. Harper, Morris, M.B., and Speicher, D.W. Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple helical bundles. J. Biol. Chem. 275 (2000) 3279–3287.
DeSilva, T. M., Peng, K.C., Speicher, K.D. and Speicher, D.W. Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides. Biochemistry 31 (1992) 10872–10878.
Mehboob, S., Jacob, J., May, M., Kotula, L., Thiyagarajan, P., Johnson, M.E. and Fung, L.W.-M. Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering. Biochemistry 42 (2003) 14702–14710.
Gaetani, M., Mootien, S., Harper, S., Gallagher, P.G. and Speicher, D.W. Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site. Blood 111 (2008) 5712–5720.
Giorgia, M., Ciancia, C.D., Gallagherb, P.G. and Morrow, J.S. Spectrin oligomerization is cooperatively coupled to membrane assembly: A linkage targeted by many hereditary hemolytic anemias? Exp. Mol. Path. 70 (2001) 215–230.
Park, S., Caffrey, M.S., Johnson, M.E. and Fung, L.W.-M. Solution structural studies on human erythrocyte alpha-spectrin tetramerization site. J. Biol. Chem. 278 (2003) 21837–21844.
Long, F., McElheny, D., Jiang, S., Park, S., Caffrey, M.S. and Fung, L.W.-M. Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin. Protein Sci. 16 (2007) 2519–2530.
Antoniou, C., Lam, V.Q. and Fung, L.W.-M. Conformational changes at the tetramerization site of erythroid alpha-spectrin upon binding beta-spectrin: a spin label EPR study. Biochemistry 47 (2008) 10765–10772.
Begg, G.E., Morris, M.B. and Ralston, G.B. Comparison of the salt-dependent self-association of brain and erythroid spectrin. Biochemistry 36 (1997) 6977–6985.
Li, Q. and Fung, L.W.-M. Structural and dynamic study of the tetramerization region of non-erythroid alpha-spectrin: a frayed helix revealed by site-directed spin labeling electron paramagnetic resonance. Biochemistry 48 (2009) 206–215.
Lusitani, D., Menhart, N., Keiderling, T.A. and Fung, L.W.-M. Ionic strength effect on the thermal unfolding of α-spectrin peptides. Biochemistry 37 (1998) 16546–16554.
Yan, Y., Winograd, E., Viel, A., Cronin, T., Harrison, S.C. and Branton, D. Crystal structure of the repetitive segments of spectrin. Science 262 (1993) 2027–2030.
Grum, V.L., Li, D., MacDonald, R.L. and Mondragón, A. Structure of two repeats of spectrin suggest models of flexibility. Cell 98 (1999) 523–535.
Conway, J.F. and Parry, D.A.D. Structural features in the heptad substructure and longer range repeats of two-stranded α-fibrous proteins. Int. J. Biol. Macromol. 12 (1990) 328–334.
Pace, C.N. and Scholtz, J.M. A helix propensity scale based on experimental studies of peptides and proteins. Biophys. J. 75 (1998) 422–427.
Kohn, W.D., Kay, C.M. and Hodges, R.S. Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils. J. Mol. Biol. 283 (1998) 993–1012.
Park, S., Johnson, M.E. and Fung, L.W.-M. Nuclear magnetic resonances studies of mutations at the tetramerization region of human alpha spectrin. Blood 100 (2002) 283–288.
Oh, Y., and Fung, L.W.-M. Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin. Cell. Mol. Biol. Lett. 12 (2007) 604–620.