Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting.
The protective antigen (PA) component of the anthrax toxin (ATx) plays an essential role in the pathogenesis of the bioterrorism bacterium Bacillus anthracis. After oligomerization on the cell surface and docking of lethal factor and/or edema factor, PA is internalized and undergoes a conformational change when exposed to the low pH of the endosome to form a membrane-penetrating pore. While the structure of the PA prepore has been determined, precise structural information regarding the pore state remains lacking. Oxidative protein footprinting (OPF) can provide dynamic structural information about a protein complex through analysis of amino acid oxidation both before and after a conformational change. In this study, PA at pH 7.5 and 5.5 was exposed to hydroxyl radicals generated by ionizing radiation. Mass spectrometry was then used to both identify and quantitate the extent of oxidation of differentially modified residues. Several residues were found to be more readily oxidized at pH 7.5, most of which clustered toward the bottom plane of the prepore heptamer. Two amino acids had greater oxidation rates at pH 5.5, both found on the outer periphery of the prepore. When the OPF results were mapped to a current computational model of the pore, the accessibilities of some residues were consistent with their modeled positions in the pore (i.e., Y688 and V619/I620), while data for other residues (W346 and M350) appeared to conflict with the model. The results from this study illustrate the utility of OPF in generating empirical structural information for yet undetermined structures and offering opportunities for refinement for models thereof.
Smedley
James G
JG
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, P.O. Box 12233, Research Triangle Park, North Carolina 27709, USA.
Sharp
Joshua S
JS
Kuhn
Jeffrey F
JF
Tomer
Kenneth B
KB
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D023302:Protein Footprinting / Q000379:methods; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin
Science. 1998 May 1;280(5364):734-7
9563949
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W375-83
17452350
Biochemistry. 2003 Nov 4;42(43):12447-54
14580189
Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13147-51
15326297
Proc Natl Acad Sci U S A. 2002 May 14;99(10):7045-8
11997437
Biochemistry. 2006 May 23;45(20):6260-6
16700537
Biochemistry. 2005 Mar 8;44(9):3166-75
15736927
Biochemistry. 1999 Aug 10;38(32):10432-41
10441138
Int J Radiat Biol. 2002 Feb;78(2):101-14
11779360
Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;17:189-98
6328915
Proc Natl Acad Sci U S A. 1982 May;79(10):3162-6
6285339
Annu Rev Biophys Biomol Struct. 2006;35:251-76
16689636
J Biol Chem. 1994 Aug 12;269(32):20607-12
8051159
Anal Biochem. 2001 Feb 15;289(2):103-15
11161303
Circ Res. 1988 Sep;63(3):628-34
3136951
J Appl Microbiol. 1999 Aug;87(2):288
10475970
Nature. 1997 Feb 27;385(6619):833-8
9039918
Rapid Commun Mass Spectrom. 2002;16(1):53-61
11754247
Mol Cell Proteomics. 2003 Oct;2(10):1120-32
12966145
Annu Rev Biochem. 2007;76:243-65
17335404
Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13278-83
16141341
J Biol Chem. 2005 Nov 25;280(47):39417-22
16183642
Anal Biochem. 2003 Feb 15;313(2):216-25
12605858
J Biol Chem. 1992 Aug 15;267(23):16396-402
1644824
Proc Natl Acad Sci U S A. 2002 May 14;99(10):7049-53
11997439
J Am Soc Mass Spectrom. 2005 Dec;16(12):2057-63
16263307
J Cell Biol. 2003 Feb 3;160(3):321-8
12551953
Nature. 2001 Nov 8;414(6860):225-9
11700562
Anal Chem. 1989 Nov 15;61(22):2504-11
2817405
Biochemistry. 2002 Feb 5;41(5):1445-50
11814336
Anal Chem. 2005 Jul 15;77(14):4549-55
16013872
Biochemistry. 2003 Oct 21;42(41):11992-2000
14556630
Biochemistry. 2002 May 7;41(18):5765-75
11980480
Biochem Biophys Res Commun. 2001 Sep 28;287(3):614-21
11563839
Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10277-81
1438214
Prog Biophys Mol Biol. 2005 May;88(1):91-142
15561302
Mol Cell Biol. 2005 Jul;25(13):5492-8
15964805
Anal Chem. 2005 Sep 15;77(18):5814-22
16159110
Biochemistry. 2003 Apr 8;42(13):3655-65
12667055
Anal Chem. 2005 May 15;77(10):3029-37
15889890
J Biomol Struct Dyn. 2004 Dec;22(3):253-65
15473701
Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5170-4
12700348
Eur J Biochem. 2001 Nov;268(21):5578-88
11683881
Anal Biochem. 2005 May 15;340(2):201-12
15840492