Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes.
The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation.
Schedin-Weiss
Sophia
S
Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden.
Richard
Benjamin
B
Olson
Steven T
ST
D000494:Allosteric Regulation; D000991:Antithrombins / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006493:Heparin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation
D000991:Antithrombins; D009844:Oligosaccharides; D006493:Heparin
EMBO J. 2006 May 3;25(9):2029-37
16619025
Adv Exp Med Biol. 1997;425:17-33
9433486
Biochemistry. 2003 Jul 15;42(27):8143-52
12846563
J Biol Chem. 1992 Jun 25;267(18):12528-38
1618758
Biochemistry. 2008 Dec 23;47(51):13610-9
19035835
J Biol Chem. 2000 Feb 11;275(6):4092-8
10660568
J Biol Chem. 1998 Jul 3;273(27):16824-7
9642241
J Biol Chem. 1981 Nov 10;256(21):11073-9
7287752
J Cell Biol. 1990 Sep;111(3):1293-304
2144002
Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):645-50
20080729
Biochemistry. 1997 Jun 3;36(22):6682-91
9184148
Trends Cardiovasc Med. 2002 Jul;12(5):198-205
12161073
Nature. 2000 Oct 19;407(6806):923-6
11057674
J Biol Chem. 2003 Apr 18;278(16):13688-95
12591924
Structure. 1994 Apr 15;2(4):257-70
8087553
Chem Rev. 2002 Dec;102(12):4751-804
12475206
Biophys J. 1999 Mar;76(3):1320-9
10049315
Biochimie. 2003 Jan-Feb;85(1-2):83-9
12765778
J Biol Chem. 2001 Apr 20;276(16):13077-86
11278457
J Biol Chem. 2002 Mar 8;277(10):8551-8
11741963
J Biol Chem. 1986 Jun 5;261(16):7507-17
2940242
FEBS Lett. 2009 Nov 3;583(21):3397-400
19818773
J Biol Chem. 1996 Nov 15;271(46):29353-8
8910598
Biochemistry. 1996 Jul 2;35(26):8495-503
8679610
FEBS Lett. 2006 Aug 21;580(19):4709-12
16884719
Biochemistry. 1998 Mar 10;37(10):3272-7
9521646
J Mol Biol. 2009 Mar 13;386(5):1278-89
19452598
Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14683-8
9405673
J Biol Chem. 1998 Mar 27;273(13):7478-87
9516447
J Biol Chem. 2001 May 4;276(18):14961-71
11278930
Biochemistry. 2003 Jul 29;42(29):8712-9
12873131
J Biol Chem. 2008 Sep 19;283(38):26137-47
18658146
Nat Struct Mol Biol. 2004 Sep;11(9):857-62
15311269
J Biol Chem. 2006 May 12;281(19):13424-13432
16517611
Nat Struct Mol Biol. 2004 Sep;11(9):863-7
15311268
J Biol Chem. 2003 Dec 19;278(51):51433-40
14532267
J Biol Chem. 1998 Sep 4;273(36):23283-9
9722560
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4808-13
10220375
J Biol Chem. 1991 Apr 5;266(10):6353-64
2007588