Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin.


Abstract

The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation. Study holds ProTherm entries: 18176, 18177, 18178, 18179, 18180, 18181, 18182, 18183 Extra Details: Amino terminal domain (residues 1-263); transition 1 anthrax toxin; lethal factor; edema factor; translocation; unfolding pathway

Submission Details

ID: fFHBYmYN3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Krantz BA;Trivedi AD;Cunningham K;Christensen KA;Collier RJ,J. Mol. Biol. (2004) Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin. PMID:15533442
Additional Information

Study Summary

Number of data points 16
Proteins Lethal factor ; Lethal factor
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: m ; Experimental Assay: dG_H2O
Libraries Mutations for sequence AGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS ; Mutations for sequence MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQRMLARYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDALLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTKLITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINS

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2L0R 2010-12-22 Conformational Dynamics of the Anthrax Lethal Factor Catalytic Center
4DV8 2012-03-14 1.63 Anthrax Lethal Factor metalloproteinase in complex with the Hydroxamic acid based small molecule PT8421
4PKW 2014-11-12 1.75 Anthrax toxin lethal factor with bound small molecule inhibitor GM6001
5D1S 2015-11-11 2.1 Anthrax toxin lethal factor with hydroxamic acid inhibitor
4PKQ 2014-11-12 2.2 Anthrax toxin lethal factor with bound zinc
5D1T 2015-11-11 2.2 Anthrax toxin lethal factor with hydroxamic acid inhibitor
4PKR 2014-11-12 2.2 Anthrax toxin lethal factor with bound small molecule inhibitor 10
4PKS 2014-11-12 2.3 Anthrax toxin lethal factor with bound small molecule inhibitor 11
1J7N 2001-11-07 2.3 Anthrax Toxin Lethal factor
1YQY 2005-05-31 2.3 Structure of B. Anthrax Lethal factor in complex with a hydroxamate inhibitor
4XM6 2015-11-11 2.35 Anthrax toxin lethal factor with ligand-induced binding pocket
4PKT 2014-11-12 2.4 Anthrax toxin lethal factor with bound small molecule inhibitor 13
4PKU 2014-11-12 2.4 Anthrax toxin lethal factor with bound small molecule inhibitor 15
4PKV 2014-11-12 2.5 Anthrax toxin lethal factor with bound small molecule inhibitor 16
4WF6 2015-09-09 2.65 Anthrax toxin lethal factor with bound small molecule inhibitor MK-31
4XM8 2015-11-11 2.7 Anthrax toxin lethal factor with ligand-induced binding pocket
1PWU 2004-02-03 2.7 Crystal Structure of Anthrax Lethal Factor complexed with (3-(N-hydroxycarboxamido)-2-isobutylpropanoyl-Trp-methylamide), a known small molecule inhibitor of matrix metalloproteases.
4XM7 2015-11-11 2.7 Anthrax toxin lethal factor with ligand-induced binding pocket
1PWW 2004-01-27 2.8 Crystal structure of Anthrax Lethal Factor active site mutant protein complexed with an optimised peptide substrate in the presence of zinc.
1PWV 2004-02-03 2.85 Crystal structure of Anthrax Lethal Factor wild-type protein complexed with an optimised peptide substrate.
5D1U 2015-11-11 2.85 Anthrax toxin lethal factor with hydroxamic acid inhibitor
1PWP 2004-01-13 2.9 Crystal Structure of the Anthrax Lethal Factor complexed with Small Molecule Inhibitor NSC 12155
3KWV 2010-11-10 3.1 Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers
1PWQ 2004-01-13 3.52 Crystal structure of Anthrax Lethal Factor complexed with Thioacetyl-Tyr-Pro-Met-Amide, a metal-chelating peptidyl small molecule inhibitor
1JKY 2001-11-07 3.9 Crystal Structure of the Anthrax Lethal Factor (LF): Wild-type LF Complexed with the N-terminal Sequence of MAPKK2

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lethal factor P15917 LEF_BACAN