Conformational changes at the tetramerization site of erythroid alpha-spectrin upon binding beta-spectrin: a spin label EPR study.
We used cysteine scanning, isothermal titration calorimetry (ITC) and spin label EPR methods to study the two regions that flank the partial domain Helix C' of the N-terminal end of alpha-spectrin (residues 14-20 and residues 44-54) in the absence and presence of a model protein of the beta-spectrin C-terminal end. In the absence of beta-spectrin, residues 14-20 and 46-52 were known to be unstructured. The EPR spectral values of the inverse line width (Delta H (-1)) and of the width between the low field peak and the central peak ( aZ) of residues in part of the first unstructured region (residues 17-20) and of most residues in the second unstructured junction region (residues 46-52) changed dramatically upon association with beta-spectrin, suggesting that the two regions undergo a conformational change, becoming more rigid and likely becoming helical. ITC results showed that three of the seven residues in the junction region (residues 46-52) were very important in its association with beta-spectrin, in the following order: L49 > G46 > K48. In general, our results suggest that any mutations that affect the propensity of helical formation in the region spanning residues 17-52 in alpha-spectrin, or that affect hydrophobic clustering and/or salt-bridge stabilization of the bundled helices, would affect spectrin tetramer formation, and may lead to blood disorders.
Antoniou
Chloe
C
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, MC 111, Chicago, Illinois 60607, USA.
Lam
Vinh Q
VQ
Fung
L W-M
LW
D000818:Animals; D002151:Calorimetry / Q000379:methods; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D013049:Spectrin
Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1667-71
8127863
Biochemistry. 2004 Jun 15;43(23):7273-87
15182173
Biophys J. 2000 Jul;79(1):526-35
10866978
Curr Opin Struct Biol. 2004 Feb;14(1):104-9
15102456
Protein Sci. 2008 Apr;17(4):601-5
18359854
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6592-6
6935670
Eur J Haematol. 2002 Feb;68(2):73-9
12038451
Biochemistry. 2005 Dec 6;44(48):15898-905
16313192
Blood. 2008 Jun 15;111(12):5712-20
18218854
J Mol Biol. 1965 May;12:88-118
14343300
Biochemistry. 1998 Nov 24;37(47):16546-54
9843421
J Biol Chem. 1996 Nov 29;271(48):30410-6
8940005
Biochemistry. 2007 Sep 25;46(38):10909-20
17713930
Biochemistry. 2001 Oct 16;40(41):12457-64
11591167
Biochemistry. 1969 Oct;8(10):4108-16
5346390
Biochemistry. 2002 Feb 5;41(5):1464-73
11814339
J Biol Chem. 2005 Jan 7;280(1):753-67
15501827
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12765-70
12239350
J Biol Chem. 1994 Oct 21;269(42):25955-8
7929303
Biophys J. 1998 Jul;75(1):422-7
9649402
Cell. 1999 Aug 20;98(4):523-35
10481916
J Mol Biol. 1982 May 5;157(1):105-32
7108955
Blood. 2002 Jul 1;100(1):283-8
12070038
Protein Sci. 2007 Nov;16(11):2519-30
17905835
Anal Biochem. 2008 May 1;376(1):160-2
18316034
Biochem J. 1998 May 15;332 ( Pt 1):81-9
9576854
Blood. 1990 Jun 1;75(11):2235-44
2346784
Nat Struct Biol. 1998 Jun;5(6):459-69
9628484
Biochemistry. 1996 Jun 18;35(24):7692-704
8672470
Curr Opin Struct Biol. 2006 Oct;16(5):644-53
16949813
Eur J Biochem. 1978 Aug 1;88(2):379-85
689023
Blood. 1986 Jan;67(1):141-51
3940543
Biochemistry. 2001 Apr 3;40(13):3828-46
11300763
Proc Natl Acad Sci U S A. 1958 Feb;44(2):98-104
16590179
Protein Sci. 2008 Feb;17(2):228-39
18096642
J Biol Chem. 2002 Jul 5;277(27):24294-8
11983696
Biochemistry. 2003 Dec 16;42(49):14702-10
14661984
Bioinformatics. 2002 Apr;18(4):617-25
12016059
Semin Hematol. 1993 Jan;30(1):21-33
8434258
Biochemistry. 1992 Nov 10;31(44):10872-8
1420200
J Clin Invest. 1990 Sep;86(3):909-16
1975598
J Biol Chem. 2003 Jun 13;278(24):21837-44
12672815
J Clin Invest. 1990 Aug;86(2):548-54
2384601
Am J Hematol. 2001 Aug;67(4):247-51
11443637
Blood Rev. 2007 Jan;21(1):1-20
16730867