Comparative analysis of the biochemical and functional properties of C-terminal domains of autotransporters.
Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.
Marín
Elvira
E
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid (UAM), Cantoblanco, Madrid 28049, Spain.
Bodelón
Gustavo
G
Fernández
Luis Ángel
LÁ
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology*; D021381:Protein Transport; D011994:Recombinant Proteins
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins
Science. 2003 Aug 22;301(5636):1099-102
12934009
Mol Microbiol. 2009 Mar;71(5):1323-32
19170888
Infect Immun. 2000 May;68(5):2720-7
10768965
Mol Microbiol. 2004 May;52(4):1069-80
15130125
Infect Immun. 2002 Nov;70(11):6355-64
12379715
J Bacteriol. 2007 Aug;189(15):5566-73
17526712
EMBO J. 1992 Jun;11(6):2327-35
1600950
Anal Biochem. 1994 Mar;217(2):220-30
8203750
Biochemistry. 2001 Nov 6;40(44):13288-301
11683639
Nat Rev Microbiol. 2009 Mar;7(3):206-14
19182809
Mol Microbiol. 2005 Nov;58(4):945-58
16262782
Nucleic Acids Res. 2003 Jul 1;31(13):3503-6
12824354
Mol Microbiol. 2000 Jul;37(2):239-53
10931321
Nat Biotechnol. 2000 Jun;18(6):661-5
10835606
J Bacteriol. 1986 Nov;168(2):595-606
3536853
Infect Immun. 2001 Nov;69(11):6769-75
11598049
Mol Microbiol. 2002 Feb;43(4):895-905
11929540
Annu Rev Microbiol. 2007;61:89-112
17506669
Infect Immun. 2001 Jan;69(1):543-6
11119549
Nucleic Acids Res. 2010 Jan;38(Database issue):D211-22
19920124
J Mol Biol. 1999 Sep 17;292(2):195-202
10493868
J Biochem. 2006 Aug;140(2):275-83
16829683
Infect Immun. 2005 Dec;73(12):8353-61
16299333
J Biol Chem. 1995 Dec 29;270(52):30874-80
8537341
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W400-4
15215419
J Mol Biol. 2010 Feb 26;396(3):627-33
20060837
J Bacteriol. 2009 Aug;191(16):5169-79
19525348
FEMS Microbiol Lett. 1998 Jun 1;163(1):65-72
9631547
Mol Membr Biol. 2006 Nov-Dec;23(6):466-74
17127619
Trends Microbiol. 2007 Oct;15(10):441-7
17935998
Mol Microbiol. 2008 Jan;67(1):188-201
18047580
J Bacteriol. 2007 Jul;189(14):5393-8
17513479
Nature. 2001 Jan 25;409(6819):529-33
11206551
J Mol Biol. 2006 Jul 21;360(4):750-9
16797588
EMBO J. 2002 May 1;21(9):2122-31
11980709
Mol Microbiol. 2007 Mar;63(5):1524-36
17302825
EMBO J. 2004 Mar 24;23(6):1257-66
15014442
Cell Microbiol. 2004 Dec;6(12):1167-83
15527496
Environ Microbiol. 2008 Mar;10(3):589-604
18237301
Vet Immunol Immunopathol. 2009 Mar 15;128(1-3):178-83
19026455
Mol Microbiol. 1994 Apr;12(2):307-19
8057855
Microbiology (Reading). 2009 Dec;155(Pt 12):3982-3991
19815580
Infect Immun. 2002 Aug;70(8):4447-61
12117956
PLoS Biol. 2006 Nov;4(11):e377
17090219
Mol Microbiol. 1999 Sep;33(6):1232-43
10510237
J Mol Biol. 2006 Mar 17;357(1):49-61
16427080
Microbiol Mol Biol Rev. 2004 Dec;68(4):692-744
15590781
Trends Biochem Sci. 2001 Apr;26(4):230-5
11295555
Nucleic Acids Res. 2003 Jul 1;31(13):3305-7
12824313
Nucleic Acids Res. 2004 May 11;32(8):2566-77
15141026
J Bacteriol. 2009 Feb;191(3):815-21
19047350
Nat Struct Mol Biol. 2007 Dec;14(12):1214-20
17994105
J Biol Chem. 2003 Dec 5;278(49):49316-22
14506253
Mol Microbiol. 1997 Feb;23(4):693-704
9157241
Nature. 1987 Jan 29-Feb 4;325(6103):458-62
3027577
Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19120-5
19850876
Infect Immun. 2005 Jul;73(7):3851-9
15972470
J Bacteriol. 1988 Mar;170(3):1245-53
3277950
J Bacteriol. 2003 Sep;185(18):5585-90
12949111
EMBO J. 1990 Jun;9(6):1991-9
2189728
J Bacteriol. 1999 Nov;181(22):7014-20
10559167
J Bacteriol. 2001 Feb;183(3):951-8
11208794
J Mol Biol. 2002 Jun 28;320(1):1-10
12079330
J Bacteriol. 1998 Sep;180(17):4452-9
9721282