title: Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy.
abstract: Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 650 East High Street, Oxford, OH, 45056, USA.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.forename: Lindsay
authors.initials: L
authors.lastname: Moritz
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.forename: Omar
authors.initials: O
authors.lastname: Sanyurah
authors.forename: Zahilyn
authors.initials: Z
authors.lastname: Roche
authors.forename: Robert
authors.initials: R
authors.lastname: McCarrick
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D013057:Spectrum Analysis; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase
references.citation: Antimicrob Agents Chemother. 1990 Aug;34(8):1590-2
references.pmid: 2121094
references.citation: Biochemistry. 2011 Nov 1;50(43):9212-24
references.pmid: 21942265
references.citation: J Biol Chem. 1999 May 28;274(22):15706-11
references.pmid: 10336469
references.citation: J Biol Chem. 2001 Nov 30;276(48):45015-23
references.pmid: 11564740
references.citation: Biochemistry. 2000 Apr 18;39(15):4288-98
references.pmid: 10757977
references.citation: J Biol Inorg Chem. 2002 Sep;7(7-8):704-12
references.pmid: 12203007
references.citation: J Am Chem Soc. 2008 Jul 2;130(26):8215-22
references.pmid: 18528987
references.citation: Biochemistry. 2000 Sep 19;39(37):11330-9
references.pmid: 10985778
references.citation: Biochemistry. 2014 Nov 25;53(46):7321-31
references.pmid: 25356958
references.citation: FEBS Lett. 2001 Aug 10;503(1):1-6
references.pmid: 11513844
references.citation: J Biol Chem. 1998 Aug 28;273(35):22402-8
references.pmid: 9712862
references.citation: Nature. 2005 Feb 10;433(7026):657-61
references.pmid: 15654328
references.citation: Clin Microbiol Infect. 2010 Dec;16(12):1699-701
references.pmid: 20874758
references.citation: Microb Drug Resist. 1996 Summer;2(2):253-6
references.pmid: 9158768
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: J Biol Chem. 2004 Sep 17;279(38):39663-70
references.pmid: 15271998
references.citation: Biochemistry. 2012 May 8;51(18):3839-47
references.pmid: 22482529
references.citation: Protein Expr Purif. 2004 Aug;36(2):272-9
references.pmid: 15249050
references.citation: Biochemistry. 1999 Nov 2;38(44):14507-14
references.pmid: 10545172
references.citation: J Mol Biol. 2005 Jan 28;345(4):785-95
references.pmid: 15588826
references.citation: Curr Top Med Chem. 2013;13(10):1242-53
references.pmid: 23647546
references.citation: Clin Microbiol Rev. 2010 Jan;23(1):160-201
references.pmid: 20065329
references.citation: J Comput Chem. 2005 Dec;26(16):1781-802
references.pmid: 16222654
references.citation: J Am Chem Soc. 2014 May 21;136(20):7273-85
references.pmid: 24754678
references.citation: Biochemistry. 1998 May 12;37(19):6791-800
references.pmid: 9578564
references.citation: Biochemistry. 2011 Oct 25;50(42):9125-34
references.pmid: 21928807
references.citation: Annu Rev Phys Chem. 2012;63:419-46
references.pmid: 22404592
references.citation: Antimicrob Agents Chemother. 1998 Apr;42(4):921-6
references.pmid: 9559809
references.citation: J Mol Biol. 1998 Nov 20;284(1):125-36
references.pmid: 9811546
references.citation: Lancet Infect Dis. 2011 May;11(5):381-93
references.pmid: 21530894
references.citation: J Antimicrob Chemother. 1996 Mar;37(3):423-31
references.pmid: 9182099
references.citation: J Med Chem. 2010 Jul 8;53(13):4862-76
references.pmid: 20527888
references.citation: Structure. 1999 Sep 15;7(9):1067-78
references.pmid: 10508780
references.citation: Biochemistry. 2005 Jan 25;44(3):1078-87
references.pmid: 15654764
references.citation: Ann N Y Acad Sci. 2013 Jan;1277:91-104
references.pmid: 23163348
references.citation: J Biol Chem. 2005 Dec 9;280(49):40668-75
references.pmid: 16227621
references.citation: Biochim Biophys Acta. 2013 Aug;1834(8):1648-59
references.pmid: 23632317
references.citation: Bioorg Med Chem Lett. 2013 Nov 1;23(21):5855-9
references.pmid: 24064498
references.citation: Biochemistry. 2006 Sep 5;45(35):10729-38
references.pmid: 16939225
references.citation: Chem Biol. 1998 Apr;5(4):185-96
references.pmid: 9545432
references.citation: Biophys J. 2010 Mar 17;98(6):L18-20
references.pmid: 20303847
references.citation: Structure. 1996 Jul 15;4(7):823-36
references.pmid: 8805566
references.citation: Biochemistry. 1999 Feb 2;38(5):1547-53
references.pmid: 9931021
references.citation: Future Med Chem. 2013 Jul;5(11):1243-63
references.pmid: 23859206
references.citation: Bioorg Med Chem Lett. 2011 Jun 1;21(11):3282-5
references.pmid: 21536436
references.citation: Biochemistry. 1999 Aug 3;38(31):10013-23
references.pmid: 10433708
references.citation: Chem Biol. 2003 Apr;10(4):319-29
references.pmid: 12725860
references.citation: Protein Sci. 2003 Jul;12(7):1368-75
references.pmid: 12824483
references.citation: J Magn Reson. 2012 Mar;216:69-77
references.pmid: 22341208
references.citation: Antimicrob Agents Chemother. 2010 Mar;54(3):969-76
references.pmid: 19995920
references.citation: Expert Opin Ther Pat. 2010 Oct;20(10):1277-93
references.pmid: 20839927
references.citation: J Magn Reson. 2013 Jul;232:53-61
references.pmid: 23694751
references.citation: J Inorg Biochem. 2014 Jul;136:40-6
references.pmid: 24742748
references.citation: Biochemistry. 2000 Nov 7;39(44):13356-64
references.pmid: 11063572