title: The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli: structural and functional studies.
abstract: Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Donata
authors.initials: D
authors.lastname: Figaj
authors.affiliation: Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk.
authors.forename: Artur
authors.initials: A
authors.lastname: Gieldon
authors.affiliation: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Sobiecka-Szkatula
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Koper
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Milena
authors.initials: M
authors.lastname: Denkiewicz
authors.affiliation: Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland, and.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Banecki
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.affiliation: Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-952 Gdansk.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Ciarkowski
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
authors.affiliation: From the Department of Biochemistry, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland, joanna.skorko-glonek@biol.ug.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; C061682:DegP protease; D012697:Serine Endopeptidases
keywords: Enzyme Mutation; Fluorescence; Molecular Biology; Molecular Modeling; Protein Structure; Serine Protease
references.citation: Biochim Biophys Acta. 2003 Jul 30;1649(2):171-82
references.pmid: 12878036
references.citation: J Biol Chem. 1997 Nov 28;272(48):29987-90
references.pmid: 9374470
references.citation: Methods Mol Biol. 2010;634:343-54
references.pmid: 20676995
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Cell Mol Life Sci. 2013 Mar;70(5):761-75
references.pmid: 22806565
references.citation: Nat Struct Mol Biol. 2010 Jul;17(7):837-43
references.pmid: 20581826
references.citation: J Chem Theory Comput. 2009 Mar 10;5(3):627-640
references.pmid: 20161452
references.citation: Proteins. 2003 Feb 15;50(3):437-50
references.pmid: 12557186
references.citation: EMBO Rep. 2010 Oct;11(10):798-804
references.pmid: 20814423
references.citation: J Mol Biol. 2001 Oct 19;313(2):417-30
references.pmid: 11800566
references.citation: Bioinformatics. 2005 Sep 1;21 Suppl 2:ii72-6
references.pmid: 16204129
references.citation: Arch Biochem Biophys. 2010 Aug 15;500(2):123-30
references.pmid: 20515644
references.citation: Proteins. 1986 Oct;1(2):109-15
references.pmid: 3130621
references.citation: Proc Natl Acad Sci U S A. 2012 May 8;109(19):7263-8
references.pmid: 22529381
references.citation: Curr Pharm Des. 2013;19(6):977-1009
references.pmid: 23016688
references.citation: J Bacteriol. 2007 Feb;189(3):706-16
references.pmid: 17122339
references.citation: Nature. 2002 Mar 28;416(6879):455-9
references.pmid: 11919638
references.citation: Proteins. 2000 Aug 15;40(3):389-408
references.pmid: 10861930
references.citation: Anal Biochem. 2010 Apr 15;399(2):196-201
references.pmid: 20074540
references.citation: Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8
references.pmid: 3156376
references.citation: J Phys Chem B. 2012 Mar 1;116(8):2365-75
references.pmid: 22260550
references.citation: J Photochem Photobiol B. 2006 Jun 1;83(3):223-36
references.pmid: 16488620
references.citation: Subcell Biochem. 2013;66:161-92
references.pmid: 23479441
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9459-64
references.pmid: 10449714
references.citation: Mol Cell. 2002 Sep;10(3):443-55
references.pmid: 12408815
references.citation: Nat Struct Mol Biol. 2010 Jul;17(7):844-52
references.pmid: 20581825
references.citation: Protein Sci. 1995 Mar;4(3):337-60
references.pmid: 7795518
references.citation: Fold Des. 1998;3(6):469-79
references.pmid: 9889162
references.citation: Gene. 1995 Sep 22;163(1):47-52
references.pmid: 7557477
references.citation: Biochim Biophys Acta. 2009 Nov;1794(11):1573-82
references.pmid: 19615474
references.citation: Cell. 2011 Apr 1;145(1):67-78
references.pmid: 21458668
references.citation: J Biol Chem. 2012 Mar 23;287(13):10115-10120
references.pmid: 22337879
references.citation: J Bacteriol. 1990 Apr;172(4):1791-7
references.pmid: 2180903
references.citation: Nature. 2008 Jun 12;453(7197):885-90
references.pmid: 18496527
references.citation: J Phys Chem B. 2007 Feb 15;111(6):1416-23
references.pmid: 17249714
references.citation: Cell Stress Chaperones. 2013 Jan;18(1):35-51
references.pmid: 22851136