Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.
The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.
Department of Biology, University of Texas at Arlington, Arlington, TX 76019; Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695.
Joseph J
JJ
Maciag
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695.
Sarah H
SH
Mackenzie
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695.
Matthew B
MB
Tucker
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695.
Joshua L
JL
Schipper
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695.
Paul
P
Swartz
Department of Biology, University of Texas at Arlington, Arlington, TX 76019; Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695; Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC 27695 clay.clark@uta.edu.
A Clay
AC
Clark
D000494:Allosteric Regulation; D000495:Allosteric Site; D019943:Amino Acid Substitution; D053148:Caspase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D021281:Quantitative Structure-Activity Relationship; D012995:Solubility; D014867:Water / Q000737:chemistry*
D014867:Water; D053148:Caspase 3
allostery; conformational selection; protein solvation; protein structure; saturation mutagenesis
Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5129-34
19251640
Biochemistry. 2006 Aug 1;45(30):9059-67
16866351
Biochemistry. 2001 Nov 27;40(47):14236-42
11714277
BMC Neurosci. 2011 Oct 12;12:102
21988729
Bioinformatics. 2013 Apr 1;29(7):845-54
23407358
J Biol Chem. 2009 May 8;284(19):12772-82
19258326
Cell. 2001 Nov 2;107(3):399-407
11701129
Biochemistry. 2001 Nov 27;40(47):14224-35
11714276
J Mol Biol. 2008 Sep 19;381(5):1157-67
18590738
Neuron. 2011 May 26;70(4):758-72
21609830
J Mol Recognit. 2003 May-Jun;16(3):121-4
12833566
Biochem J. 2009 Dec 10;424(3):335-45
19788411
J Biol Chem. 2006 Feb 10;281(6):3254-60
16339151
J Biol Chem. 2012 Oct 19;287(43):36000-11
22891250
Chem Rev. 2016 Jun 8;116(11):6666-706
26750439
Biochem J. 2004 Dec 15;384(Pt 3):515-25
15312047
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Aug 1;67(Pt 8):842-50
21821879
Cell. 2001 Mar 9;104(5):769-80
11257230
Cell. 2001 Mar 9;104(5):791-800
11257232
Cell. 2009 Sep 4;138(5):838-54
19737514
Proc Natl Acad Sci U S A. 2013 May 21;110(21):8477-82
23650375
Cold Spring Harb Perspect Biol. 2013 Jun 01;5(6):null
23732469
J Biol Chem. 2005 Aug 26;280(34):30263-72
15994297
Methods Enzymol. 2014;544:215-49
24974292
Biochem J. 2004 Dec 1;384(Pt 2):201-32
15450003
Structure. 2004 Aug;12(8):1361-71
15296730
Science. 1992 May 1;256(5057):655-9
1585178
Proc Natl Acad Sci U S A. 2006 May 16;103(20):7595-600
16682620
ACS Chem Biol. 2013 Jul 19;8(7):1558-66
23614665
Biochem Pharmacol. 2003 Oct 15;66(8):1453-8
14555221
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
20124702
Q Rev Biophys. 2010 Aug;43(3):295-332
20819242
Structure. 2012 Apr 4;20(4):742-51
22483120
J Exp Med. 2004 Feb 16;199(4):449-58
14970175
J Med Chem. 2006 Sep 21;49(19):5728-49
16970398
Curr Opin Cell Biol. 2003 Dec;15(6):725-31
14644197
Biochemistry. 2006 Nov 7;45(44):13249-63
17073446
Arch Biochem Biophys. 2011 Apr 1;508(1):31-8
21266160
Protein Sci. 2009 Jul;18(7):1459-68
19530232
Biosci Rep. 2012 Aug;32(4):401-11
22607239
J Biol Chem. 2012 May 4;287(19):15371-9
22433863
Cell Death Differ. 2010 Jul;17(7):1104-14
19960023
Nat Biotechnol. 2003 Mar;21(3):308-14
12563278
J Mol Biol. 2014 Feb 6;426(3):611-29
24189050
Biophys J. 2003 Jan;84(1):564-70
12524309
Nature. 2014 Apr 17;508(7496):331-9
24740064
Annu Rev Biophys Biomol Struct. 2006;35:389-415
16689642
Cell. 2012 Jul 20;150(2):426-40
22817901
J Mol Biol. 2006 Jun 23;359(5):1378-88
16787777
Mol Cell Proteomics. 2013 Dec;12(12):3719-31
24023391
Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12461-6
15314233
Biochemistry. 2014 Dec 9;53(48):7582-95
25343534
Biochemistry. 2003 Oct 28;42(42):12311-20
14567692
Annu Rev Biochem. 2011;80:1055-87
21456965
EMBO J. 2011 Jan 19;30(2):379-94
21157428
Biochem Pharmacol. 2008 Jun 1;75(11):2234-44
18395187
Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14790-5
11752425
Adv Exp Med Biol. 2012;747:55-73
22949111
J Biol Chem. 2005 Apr 29;280(17):17371-9
15716280
Biochemistry. 2003 Oct 28;42(42):12298-310
14567691
Structure. 2007 May;15(5):625-36
17502107
Biochemistry. 2007 Jul 3;46(26):7852-64
17555301