Structural and mutational analysis of functional differentiation between synaptotagmins-1 and -7.
Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 A resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.
Xue
Mingshan
M
Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.
Craig
Timothy K
TK
Shin
Ok-Ho
OH
Li
Liyi
L
Brautigam
Chad A
CA
Tomchick
Diana R
DR
Südhof
Thomas C
TC
Rosenmund
Christian
C
Rizo
Josep
J
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D050863:Synaptotagmin I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D050857:Synaptotagmins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D018377:Neurotransmitter Agents; D050863:Synaptotagmin I; D050857:Synaptotagmins; D002118:Calcium
Cell. 1995 Mar 24;80(6):929-38
7697723
EMBO J. 2005 Aug 17;24(16):2839-50
16052212
Nat Struct Mol Biol. 2008 Jul;15(7):665-74
18618940
Nat Struct Mol Biol. 2008 Nov;15(11):1160-8
18953334
Nat Struct Mol Biol. 2010 Mar;17(3):325-31
20173762
J Mol Biol. 2007 Mar 30;367(3):848-63
17320903
Annu Rev Biochem. 2008;77:615-41
18275379
Science. 2010 May 7;328(5979):760-3
20448186
J Biol Chem. 2008 Aug 1;283(31):21799-807
18508778
EMBO J. 1998 Jul 15;17(14):3921-30
9670009
Dev Biol. 2001 Jul 15;235(2):521-9
11437455
Nat Struct Mol Biol. 2006 Apr;13(4):323-30
16565726
Cell. 2009 Sep 4;138(5):935-46
19716167
Nat Rev Mol Cell Biol. 2006 Sep;7(9):631-43
16912714
Nat Struct Mol Biol. 2010 Mar;17(3):318-24
20173763
Biofactors. 2010 Jan-Feb;36(1):1-7
20049899
EMBO J. 1999 Nov 15;18(22):6329-38
10562545
Cell. 1994 Nov 18;79(4):717-27
7954835
Biochemistry. 2001 May 22;40(20):5854-60
11352720
Nature. 2002 Jul 18;418(6895):340-4
12110842
J Biol Chem. 1998 May 29;273(22):13995-4001
9593749
Biochem J. 2004 Mar 1;378(Pt 2):681-6
14713287
Neuron. 2001 Dec 20;32(6):1057-69
11754837
Nature. 2001 Mar 1;410(6824):41-9
11242035
Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5210-4
15793006
Methods Enzymol. 1997;276:307-26
27754618
PLoS Biol. 2006 Jul;4(7):e192
16732694
Science. 2002 Feb 1;295(5556):868-72
11786607
Structure. 1998 Nov 15;6(11):1395-405
9817842
Neuron. 2004 Jan 8;41(1):85-99
14715137
J Biol Chem. 2002 Mar 8;277(10):7629-32
11739399
Science. 1996 Jul 12;273(5272):248-51
8662510
Acta Crystallogr D Biol Crystallogr. 2003 Jul;59(Pt 7):1131-7
12832755
Nat Struct Mol Biol. 2006 Mar;13(3):209-17
16491093
Protein Sci. 2004 Oct;13(10):2665-72
15340165
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3992-7
18308938
Science. 2009 Jan 23;323(5913):474-7
19164740
Cell. 2009 Aug 21;138(4):709-21
19703397
EMBO J. 2006 May 17;25(10):2039-50
16642042
Neuron. 2003 Jul 17;39(2):299-308
12873386
Nat Struct Mol Biol. 2007 Oct;14(10):949-58
17828276
J Biol Chem. 1998 Jun 26;273(26):15879-82
9632630
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18664-9
16352718
J Neurosci. 2004 Sep 29;24(39):8542-50
15456828
J Neurosci. 2002 Oct 1;22(19):8438-46
12351718
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
20124702
Nat Struct Mol Biol. 2004 Sep;11(9):844-9
15311271
Neuron. 2007 May 24;54(4):567-81
17521570
Nat Cell Biol. 1999 Jun;1(2):106-12
10559882
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3986-91
18308933
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3998-4003
18308932
J Biol Chem. 2004 Mar 26;279(13):12574-9
14709554
Nat Struct Mol Biol. 2004 Jan;11(1):45-53
14718922
J Cell Sci. 2007 Feb 15;120(Pt 4):617-27
17264148
Trends Cell Biol. 2006 Jul;16(7):339-50
16698267
Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7875-80
18505837
Neuron. 2001 May;30(2):459-73
11395007
Cell. 2006 Sep 22;126(6):1175-87
16990140
Biophys J. 2005 Jul;89(1):690-702
15821166
Science. 2008 Sep 12;321(5895):1507-10
18703708
Biochemistry. 1998 Nov 17;37(46):16106-15
9819203
Biochemistry. 1998 Sep 8;37(36):12395-403
9730811