title: Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.
abstract: Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Barnard
authors.forename: James
authors.initials: J
authors.lastname: Gumbart
authors.forename: Janine H
authors.initials: JH
authors.lastname: Peterson
authors.forename: Nicholas
authors.initials: N
authors.lastname: Noinaj
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Easley
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dautin
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Kuszak
authors.forename: Emad
authors.initials: E
authors.lastname: Tajkhorshid
authors.forename: Harris D
authors.initials: HD
authors.lastname: Bernstein
authors.forename: Susan K
authors.initials: SK
authors.lastname: Buchanan
mesh_terms: D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003500:Cyclization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D017434:Protein Structure, Tertiary; D011522:Protons; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011522:Protons; D014867:Water; D018698:Glutamic Acid; D001216:Asparagine; C107189:EspP protein, E coli; D012697:Serine Endopeptidases
references.citation: J Mol Biol. 2009 Nov 13;393(5):1129-42
references.pmid: 19747487
references.citation: Trends Biotechnol. 1994 Oct;12(10):420-6
references.pmid: 7765388
references.citation: Mol Microbiol. 2009 Mar;71(5):1323-32
references.pmid: 19170888
references.citation: EMBO J. 2006 Jun 7;25(11):2297-304
references.pmid: 16688217
references.citation: J Mol Biol. 2010 Feb 26;396(3):627-33
references.pmid: 20060837
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: J Phys Chem B. 1998 Apr 30;102(18):3586-616
references.pmid: 24889800
references.citation: Mol Microbiol. 1995 Nov;18(3):413-23
references.pmid: 8748026
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 4;102(1):221-6
references.pmid: 15615856
references.citation: EMBO J. 2004 Mar 24;23(6):1257-66
references.pmid: 15014442
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: J Biol Chem. 1987 Jan 15;262(2):785-94
references.pmid: 3805008
references.citation: Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6572-6
references.pmid: 7604035
references.citation: J Biol Chem. 2004 Jul 23;279(30):31495-504
references.pmid: 15151995
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Biochem Biophys Res Commun. 1990 Mar 16;167(2):711-5
references.pmid: 2182019
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):355-67
references.pmid: 21460454
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: Annu Rev Microbiol. 2007;61:89-112
references.pmid: 17506669
references.citation: BMC Struct Biol. 2011 Jan 26;11:6
references.pmid: 21269479
references.citation: J Mol Biol. 2010 Sep 17;402(2):475-89
references.pmid: 20655928
references.citation: Proteins. 2003 Feb 15;50(3):437-50
references.pmid: 12557186
references.citation: J Phys Chem B. 2009 Apr 23;113(16):5607-16
references.pmid: 19326906
references.citation: Mol Microbiol. 2000 Jul;37(2):424-32
references.pmid: 10931336
references.citation: J Bacteriol. 2011 Aug;193(15):3748-56
references.pmid: 21642456
references.citation: Mol Membr Biol. 2006 Nov-Dec;23(6):499-508
references.pmid: 17127622
references.citation: J Comput Chem. 2005 Dec;26(16):1781-802
references.pmid: 16222654
references.citation: Microbiology (Reading). 2010 Sep;156(Pt 9):2587-2596
references.pmid: 20616105
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
references.pmid: 11517324
references.citation: Toxins (Basel). 2010 Jun;2(6):1179-206
references.pmid: 22069633
references.citation: Mol Microbiol. 2005 Nov;58(4):945-58
references.pmid: 16262782
references.citation: Biochem J. 2011 May 1;435(3):577-87
references.pmid: 21306302
references.citation: EMBO J. 2007 Apr 4;26(7):1942-52
references.pmid: 17347646
references.citation: Infect Immun. 2010 Aug;78(8):3516-28
references.pmid: 20515934
references.citation: Nat Struct Mol Biol. 2007 Dec;14(12):1214-20
references.pmid: 17994105
references.citation: Biochim Biophys Acta. 2002 May 3;1562(1-2):6-31
references.pmid: 11988218
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: J Mol Biol. 2010 Oct 1;402(4):645-56
references.pmid: 20615416
references.citation: Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19120-5
references.pmid: 19850876