Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase.
Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.
Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH 45221, USA.
Andrea
A
Kravats
Manori
M
Jayasinghe
George
G
Stan
D000494:Allosteric Regulation / Q000502:physiology; D049071:Endopeptidase Clp / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology
C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D049071:Endopeptidase Clp
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4030-5
8633011
Mol Cell. 2001 Mar;7(3):627-37
11463387
Annu Int Conf IEEE Eng Med Biol Soc. 2007;2007:1213-6
18002181
Trends Biochem Sci. 1996 Aug;21(8):289-96
8772382
Mol Cell. 2000 Apr;5(4):639-48
10882100
J Comput Chem. 2009 Jul 30;30(10):1545-614
19444816
Biophys J. 2004 Jul;87(1):562-73
15240489
Genes Dev. 1998 May 1;12(9):1338-47
9573050
Proc Natl Acad Sci U S A. 2007 May 22;104(21):8803-8
17496143
Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7184-9
10377389
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8898-903
10922052
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6521-6
10823892
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10584-9
11535833
J Cell Biol. 2000 Jul 10;150(1):F13-9
10893253
Nature. 2005 Oct 20;437(7062):1115-20
16237435
J Biol Chem. 1999 Oct 1;274(40):28083-6
10497158
J Biol Chem. 2001 Aug 3;276(31):29420-9
11346657
J Mol Biol. 1999 Feb 19;286(2):553-61
9973570
Science. 2004 Mar 12;303(5664):1674-8
15017000
Nat Rev Mol Cell Biol. 2005 Jul;6(7):519-29
16072036
Genome Res. 1999 Jan;9(1):27-43
9927482
EMBO J. 1982;1(8):945-51
6329717
Biophys J. 2006 Sep 1;91(5):L51-3
16815903
Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20268-73
18079292
J Biol Chem. 1987 Apr 5;262(10):4477-85
3549708
Nature. 1999 Sep 2;401(6748):90-3
10485712
Nature. 2000 Feb 17;403(6771):800-5
10693812
J Mol Biol. 2009 Jul 17;390(3):443-56
19426737
Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8502-7
9671707
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18237-42
19828442
Trends Biochem Sci. 2006 Dec;31(12):647-53
17074491
Bioinformatics. 2007 Jul 1;23(13):i240-8
17646302
Cell. 2005 Jul 1;121(7):1017-27
15989952
Structure. 2001 Nov;9(11):1107-16
11709174
J Biol Chem. 2003 Dec 12;278(50):50664-70
14514695
Nat Struct Biol. 1997 Feb;4(2):133-9
9033594
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4328-33
11287666
J Biol Chem. 2005 Dec 9;280(49):40838-44
16207718
J Struct Biol. 2006 Oct;156(1):230-43
16765605
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8892-7
10922051
Cell. 2005 Jul 1;121(7):1029-41
15989953
Annu Rev Cell Dev Biol. 2003;19:565-87
14570582
Protein Sci. 1992 Mar;1(3):363-9
1363913
Protein Sci. 2001 Mar;10(3):551-9
11344323
Genes Cells. 2001 Jul;6(7):575-97
11473577
Mol Cell. 2003 Mar;11(3):671-83
12667450
Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13732-7
10570141
Curr Opin Struct Biol. 2008 Feb;18(1):43-51
18276129
Biopolymers. 1992 Jun;32(6):695-709
1643270
Curr Opin Struct Biol. 2002 Dec;12(6):746-53
12504679
J Mol Biol. 1996 Oct 25;263(2):323-43
8913310
J Chem Phys. 2005 Sep 22;123(12):124903
16392523
J Biol Chem. 2002 Nov 29;277(48):46743-52
12205096
Nat Struct Mol Biol. 2008 Feb;15(2):139-45
18223658