A single EF-hand isolated from STIM1 forms dimer in the absence and presence of Ca2+.
Stromal interaction molecule 1 (STIM1) is responsible for activating the Ca(2+) release-activated Ca(2+) (CRAC) channel by first sensing the changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) via its luminal canonical EF-hand motif and subsequently oligomerizing to interact with the CRAC channel pore-forming subunit Orai1. In this work, we applied a grafting approach to obtain the intrinsic metal-binding affinity of the isolated EF-hand of STIM1, and further investigated its oligomeric state using pulsed-field gradient NMR and size-exclusion chromatography. The canonical EF-hand bound Ca(2+) with a dissociation constant at a level comparable with [Ca(2+)](ER) (512 +/- 15 microm). The binding of Ca(2+) resulted in a more compact conformation of the engineered protein. Our results also showed that D to A mutations at Ca(2+)-coordinating loop positions 1 and 3 of the EF-hand from STIM1 led to a 15-fold decrease in the metal-binding affinity, which explains why this mutant was insensitive to changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) and resulted in constitutive punctae formation and Ca(2+) influx. In addition, the grafted single EF-hand motif formed a dimer regardless of the presence of Ca(2+), which conforms to the EF-hand paring paradigm. These data indicate that the STIM1 canonical EF-hand motif tends to dimerize for functionality in solution and is responsible for sensing changes in [Ca(2+)](ER).
Huang
Yun
Y
Department of Chemistry, Center for Drug Design and Advanced Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
Zhou
Yubin
Y
Wong
Hing-Cheung
HC
Chen
Yanyi
Y
Chen
Yan
Y
Wang
Siming
S
Castiblanco
Adriana
A
Liu
Aimin
A
Yang
Jenny J
JJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D066298:In Vitro Techniques; D028581:Lanthanoid Series Elements / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000071737:Stromal Interaction Molecule 1
D028581:Lanthanoid Series Elements; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium
Proteins. 2002 May 15;47(3):323-33
11948786
Cell. 2008 Oct 3;135(1):110-22
18854159
Protein Eng. 2001 Dec;14(12):1001-13
11809931
Structure. 1994 Aug 15;2(8):755-66
7994575
J Biol Chem. 2007 Jun 29;282(26):19000-10
17478419
Biochemistry. 1997 Jan 28;36(4):680-7
9020765
J Biol Chem. 2006 Nov 24;281(47):35855-62
17020874
J Mol Biol. 1975 Jan 15;91(2):201-25
1237625
J Cell Biol. 2005 May 9;169(3):435-45
15866891
J Virol. 2007 Jul;81(14):7517-28
17475644
Biochim Biophys Acta. 2002 Jul 29;1598(1-2):80-7
12147347
Nature. 1992 Nov 19;360(6401):232-9
1279440
J Biol Chem. 2009 Jan 9;284(2):728-32
19019825
Annu Rev Biophys Biomol Struct. 2000;29:291-325
10940251
Biochem Soc Trans. 1993 Nov;21(4):947-52
7907561
Cell Calcium. 2003 Aug;34(2):109-19
12810053
Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4040-5
16537481
Proteins. 2006 Nov 15;65(3):643-55
16981205
J Am Chem Soc. 2005 Mar 23;127(11):3743-50
15771508
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3693-7
17360414
Curr Biol. 2005 Jul 12;15(13):1235-41
16005298
Biochemistry. 1999 Dec 14;38(50):16424-31
10600103
Nat Cell Biol. 2009 Mar;11(3):337-43
19182790
J Magn Reson B. 1995 Jun;107(3):260-6
7788099
Nature. 2005 Oct 6;437(7060):902-5
16208375
Arch Biochem Biophys. 1999 Dec 1;372(1):80-8
10562419
J Theor Biol. 1999 Jul 21;199(2):199-211
10395814
Biometals. 1998 Dec;11(4):277-95
10191494
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W668-73
15215473
Biochemistry. 1992 Oct 13;31(40):9572-80
1390738