Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling.
The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin--lethal factor (LF) and edema factor (EF)--across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1' of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.
Department of Microbiology and Molecular Genetics and Medicine, Harvard Medical School, Boston, MA 02115, USA.
Laura D
LD
Jennings-Antipov
Likai
L
Song
R John
RJ
Collier
D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008051:Lipid Bilayers; D008958:Models, Molecular; D013113:Spin Labels
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008051:Lipid Bilayers; D013113:Spin Labels; C030325:anthrax toxin
J Biol Chem. 2006 Jan 20;281(3):1630-5
16293620
Nature. 1997 Feb 27;385(6619):833-8
9039918
ACS Chem Biol. 2010 Apr 16;5(4):359-64
20180595
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13803-8
14623961
EMBO J. 2005 Mar 9;24(5):929-41
15719022
Proc Natl Acad Sci U S A. 2004 Nov 30;101(48):16756-61
15548616
Proc Natl Acad Sci U S A. 2002 May 14;99(10):7049-53
11997439
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3453-7
20142512
Biochemistry. 1999 Aug 10;38(32):10432-41
10441138
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10277-81
1438214
J Mol Biol. 2009 Sep 25;392(3):614-29
19627991
Protein Sci. 2009 Sep;18(9):1882-95
19609933
Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5170-4
12700348
Proc Natl Acad Sci U S A. 1982 May;79(10):3162-6
6285339
Mol Aspects Med. 2009 Dec;30(6):413-22
19563824
Nat Struct Mol Biol. 2008 Jul;15(7):754-60
18568038
Nat Struct Mol Biol. 2010 Nov;17(11):1383-90
21037566
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8239-43
7667275
Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13147-51
15326297
Biochemistry. 2002 Jan 22;41(3):1079-82
11790132
Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16409-14
16251269
J Mol Biol. 2004 Nov 26;344(3):739-56
15533442
Curr Opin Struct Biol. 2006 Oct;16(5):644-53
16949813
Annu Rev Biochem. 2007;76:243-65
17335404
Protein Sci. 2009 Feb;18(2):348-58
19165720
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4346-51
18334631
Biophys J. 2004 Dec;87(6):3842-9
15377524
J Cell Biol. 2003 Feb 3;160(3):321-8
12551953
Nature. 2001 Nov 8;414(6860):225-9
11700562
Nature. 2001 Nov 8;414(6860):229-33
11700563
Proc Natl Acad Sci U S A. 1989 Apr;86(7):2209-13
2467303
J Biol Chem. 2002 Jan 25;277(4):3006-10
11714723
J Appl Microbiol. 1999 Aug;87(2):283
10475967
Science. 2005 Jul 29;309(5735):777-81
16051798
J Biol Chem. 2010 Mar 12;285(11):8130-7
20061382
Nat Struct Biol. 2000 Sep;7(9):735-9
10966640
Mol Microbiol. 1994 Sep;13(6):1093-100
7854123