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 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.
Key words: Erythroid spectrin, Tetramerization, G46, mutation, ITC
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Abbreviations
- 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
- Tm
temperature with 50% thermal unfolding
- Umid
urea concentration with 50% unfolding
References
- 1.Bennett V., Healy J. Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin. Trends Mol. Med. 2008;14:28–36. doi: 10.1016/j.molmed.2007.11.005. [DOI] [PubMed] [Google Scholar]
- 2.Mohandas N., An X. New insights into function of red cell membrane proteins and their interaction with spectrin-based membrane skeleton. Transfus. Clin. Biol. 2006;13:29–30. doi: 10.1016/j.tracli.2006.02.017. [DOI] [PubMed] [Google Scholar]
- 3.Elgsaeter A., Stokke B.T., Mikkelsen A., Branton D. The molecular basis of erythrocyte shape. Science. 1986;234:1217–1223. doi: 10.1126/science.3775380. [DOI] [PubMed] [Google Scholar]
- 4.Begg G.E., Harper S.L., Morris M.B., Speicher D.W. Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple helical bundles. J. Biol. Chem. 2000;275:3279–3287. doi: 10.1074/jbc.275.5.3279. [DOI] [PubMed] [Google Scholar]
- 5.DeSilva T. M., Peng K.C., Speicher K.D., Speicher D.W. Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides. Biochemistry. 1992;31:10872–10878. doi: 10.1021/bi00159a030. [DOI] [PubMed] [Google Scholar]
- 6.Mehboob S., Jacob J., May M., Kotula L., Thiyagarajan P., Johnson M.E., Fung L.W.-M. Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering. Biochemistry. 2003;42:14702–14710. doi: 10.1021/bi0353833. [DOI] [PubMed] [Google Scholar]
- 7.Gaetani M., Mootien S., Harper S., Gallagher P.G., Speicher D.W. Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site. Blood. 2008;111:5712–5720. doi: 10.1182/blood-2007-11-122457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Giorgia M., Ciancia C.D., Gallagherb P.G., Morrow J.S. Spectrin oligomerization is cooperatively coupled to membrane assembly: A linkage targeted by many hereditary hemolytic anemias? Exp. Mol. Path. 2001;70:215–230. doi: 10.1006/exmp.2001.2377. [DOI] [PubMed] [Google Scholar]
- 9.Park S., Caffrey M.S., Johnson M.E., Fung L.W.-M. Solution structural studies on human erythrocyte alpha-spectrin tetramerization site. J. Biol. Chem. 2003;278:21837–21844. doi: 10.1074/jbc.M300617200. [DOI] [PubMed] [Google Scholar]
- 10.Long F., McElheny D., Jiang S., Park S., Caffrey M.S., Fung L.W.-M. Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin. Protein Sci. 2007;16:2519–2530. doi: 10.1110/ps.073115307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Antoniou C., Lam V.Q., Fung L.W.-M. Conformational changes at the tetramerization site of erythroid alpha-spectrin upon binding beta-spectrin: a spin label EPR study. Biochemistry. 2008;47:10765–10772. doi: 10.1021/bi800840p. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Begg G.E., Morris M.B., Ralston G.B. Comparison of the salt-dependent self-association of brain and erythroid spectrin. Biochemistry. 1997;36:6977–6985. doi: 10.1021/bi970186n. [DOI] [PubMed] [Google Scholar]
- 13.Li Q., 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. 2009;48:206–215. doi: 10.1021/bi8013032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Lusitani D., Menhart N., Keiderling T.A., Fung L.W.-M. Ionic strength effect on the thermal unfolding of α-spectrin peptides. Biochemistry. 1998;37:16546–16554. doi: 10.1021/bi9811462. [DOI] [PubMed] [Google Scholar]
- 15.Yan Y., Winograd E., Viel A., Cronin T., Harrison S.C., Branton D. Crystal structure of the repetitive segments of spectrin. Science. 1993;262:2027–2030. doi: 10.1126/science.8266097. [DOI] [PubMed] [Google Scholar]
- 16.Grum V.L., Li D., MacDonald R.L., Mondragón A. Structure of two repeats of spectrin suggest models of flexibility. Cell. 1999;98:523–535. doi: 10.1016/s0092-8674(00)81980-7. [DOI] [PubMed] [Google Scholar]
- 17.Conway J.F., Parry D.A.D. Structural features in the heptad substructure and longer range repeats of two-stranded α-fibrous proteins. Int. J. Biol. Macromol. 1990;12:328–334. doi: 10.1016/0141-8130(90)90023-4. [DOI] [PubMed] [Google Scholar]
- 18.Pace C.N., Scholtz J.M. A helix propensity scale based on experimental studies of peptides and proteins. Biophys. J. 1998;75:422–427. doi: 10.1016/s0006-3495(98)77529-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kohn W.D., Kay C.M., Hodges R.S. Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils. J. Mol. Biol. 1998;283:993–1012. doi: 10.1006/jmbi.1998.2125. [DOI] [PubMed] [Google Scholar]
- 20.Park S., Johnson M.E., Fung L.W.-M. Nuclear magnetic resonances studies of mutations at the tetramerization region of human alpha spectrin. Blood. 2002;100:283–288. doi: 10.1182/blood.v100.1.283. [DOI] [PubMed] [Google Scholar]
- 21.Oh Y., Fung L.W.-M. Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin. Cell. Mol. Biol. Lett. 2007;12:604–620. doi: 10.2478/s11658-007-0028-8. [DOI] [PMC free article] [PubMed] [Google Scholar]