title: Mechanism of substrate translocation by a ring-shaped ATPase motor at millisecond resolution.
abstract: Ring-shaped, hexameric ATPase motors fulfill key functions in cellular processes, such as genome replication, transcription, or protein degradation, by translocating a long substrate through their central pore powered by ATP hydrolysis. Despite intense research efforts, the atomic-level mechanism transmitting chemical energy from hydrolysis into mechanical force that translocates the substrate is still unclear. Here we employ all-atom molecular dynamics simulations combined with advanced path sampling techniques and milestoning analysis to characterize how mRNA substrate is translocated by an exemplary homohexameric motor, the transcription termination factor Rho. We find that the release of hydrolysis product (ADP + Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition, the time scale seen also in experiment. The calculated free energy profiles and kinetics show that Rho unidirectionally translocates the single-stranded RNA substrate via a population shift of the conformational states of Rho; upon hydrolysis product release, the most favorable conformation shifts from the pretranslocation state to the post-translocation state. Via two previously unidentified intermediate states, the RNA chain is seen to be pulled by six K326 side chains, whose motions are induced by highly coordinated relative translation and rotation of Rho's six subunits. The present study not only reveals in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provides an effective approach to identify allosteric sites of multimeric enzymes in general.
authors: Center for Biophysics and Computational Biology and Beckman Institute for Advanced Science and Technology and ‡Department of Physics, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors: Wen
authors: W
authors: Ma
authors: Klaus
authors: K
authors: Schulten
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000494:Allosteric Regulation; D001692:Biological Transport; D006868:Hydrolysis; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012399:Rotation; D013816:Thermodynamics; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D012313:RNA; D000251:Adenosine Triphosphatases; D020741:rho GTP-Binding Proteins
references: Structure. 2012 Apr 4;20(4):618-27
references: 22483109
references: J Phys Chem B. 2008 Mar 20;112(11):3432-40
references: 18290641
references: Cell. 2012 Oct 12;151(2):267-77
references: 23022319
references: Nature. 1994 Aug 25;370(6491):621-8
references: 8065448
references: Proc Natl Acad Sci U S A. 2007 May 22;104(21):8803-8
references: 17496143
references: Cell Rep. 2014 Mar 27;6(6):1037-1045
references: 24630993
references: Mol Cell. 2006 Jun 9;22(5):611-21
references: 16762834
references: Cancer Res. 2007 Nov 15;67(22):10627-30
references: 18006801
references: Nat Commun. 2011;2:233
references: 21407199
references: Angew Chem Int Ed Engl. 1998 Sep 18;37(17):2308-2319
references: 29710950
references: J Am Chem Soc. 2014 May 14;136(19):6960-8
references: 24798048
references: Nat Struct Mol Biol. 2009 Dec;16(12):1309-16
references: 19915588
references: Nat Rev Microbiol. 2007 Nov;5(11):892-9
references: 17938630
references: Cell. 2003 Jul 11;114(1):135-46
references: 12859904
references: Philos Trans R Soc Lond B Biol Sci. 2012 Dec 24;368(1611):20120023
references: 23267177
references: Nat Chem Biol. 2009 Aug;5(8):593-9
references: 19561621
references: Biochim Biophys Acta. 1993 Jan 8;1140(3):215-50
references: 8417777
references: J Biol Chem. 2013 Nov 8;288(45):32700-32707
references: 24089518
references: Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):4870-5
references: 16551743
references: J Biol Chem. 2002 Aug 16;277(33):30023-30
references: 12034708
references: Curr Opin Struct Biol. 2011 Apr;21(2):240-8
references: 21282052
references: Comput Phys Commun. 2014 Mar;185(3):908-916
references: 24944348
references: J Chem Theory Comput. 2009 Aug 31;5(10):2589-2594
references: 20354583
references: J Chem Phys. 2004 Jun 15;120(23):10880-9
references: 15268118
references: J Comput Chem. 2005 Dec;26(16):1781-802
references: 16222654
references: Curr Opin Microbiol. 2013 Apr;16(2):118-24
references: 23347833
references: Nat Struct Biol. 2002 Mar;9(3):198-202
references: 11836535
references: PLoS Comput Biol. 2007 Feb 16;3(2):e23
references: 17305418
references: Cell. 2009 Oct 30;139(3):523-34
references: 19879839
references: Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15431-6
references: 20716688
references: Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):874-9
references: 12552084
references: J Mol Biol. 1995 Dec 15;254(5):815-37
references: 7500353
references: J Am Chem Soc. 2012 Jan 11;134(1):623-30
references: 22098642
references: Biophys J. 2010 Apr 21;98(8):1449-57
references: 20409463
references: Nature. 2001 Apr 19;410(6831):898-904
references: 11309608
references: J Chem Phys. 2008 Nov 7;129(17):174102
references: 19045328
references: Science. 2011 Oct 28;334(6055):517-20
references: 22034434
references: Nat Commun. 2014 Mar 03;5:3397
references: 24584478
references: J Mol Biol. 2001 Dec 14;314(5):1007-15
references: 11743718
references: Cell Cycle. 2014;13(10):1509-10
references: 24755939
references: Curr Opin Struct Biol. 2011 Apr;21(2):167-72
references: 21333527
references: J Chem Phys. 2009 May 21;130(19):194101
references: 19466815
references: Genes Dev. 2013 Nov 15;27(22):2500-11
references: 24240239
references: J Mol Biol. 2012 Nov 9;423(5):664-76
references: 22885804
references: J Clin Invest. 2014 Jan;124(1):6-12
references: 24382383
references: Top Curr Chem. 2013;337:139-64
references: 23468286
references: Biochemistry. 2002 Aug 6;41(31):10021-5
references: 12146966
references: Proc Natl Acad Sci U S A. 2006 Dec 12;103(50):18939-44
references: 17135353
references: J Biol Chem. 2009 Dec 4;284(49):33773-80
references: 19837672
references: Blood. 2012 Apr 5;119(14):3321-9
references: 22323447
references: J Phys Chem B. 2012 Jul 26;116(29):8573-83
references: 22616575
references: Biophys J. 2014 May 6;106(9):1844-58
references: 24806916
references: Nature. 2013 Jan 31;493(7434):703-7
references: 23334411
references: Nature. 2013 May 30;497(7451):643-6
references: 23719463
references: Biochim Biophys Acta. 2002 Sep 13;1577(2):251-260
references: 12213656
references: Cell Rep. 2014 Mar 27;6(6):1129-1138
references: 24630996
references: Biomed Res Int. 2014;2014:549719
references: 25243149
references: Nature. 2006 Jul 20;442(7100):270-5
references: 16855583
references: Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19814-9
references: 21041674
references: Nature. 2010 Jan 14;463(7278):245-9
references: 20075920
references: Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5001-5
references: 20194770
references: EMBO J. 2004 Jul 21;23(14):2734-44
references: 15229653
references: Proc Natl Acad Sci U S A. 2009 May 5;106(18):7449-54
references: 19383795
references: Proc Natl Acad Sci U S A. 2013 Oct 8;110(41):16468-73
references: 24062450
references: J Mol Biol. 2011 Jan 14;405(2):497-518
references: 21059356
references: IUBMB Life. 2014 Sep;66(9):624-30
references: 25229752
references: Structure. 2011 Apr 13;19(4):588-98
references: 21481781
references: J Am Chem Soc. 2012 May 23;134(20):8447-54
references: 22548707
references: J Chem Theory Comput. 2010;6(6):1805-1817
references: 20596240
references: J Chem Phys. 2006 Nov 7;125(17):174108
references: 17100430