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. 2005 Nov 8;102(45):16409-14.
doi: 10.1073/pnas.0508259102. Epub 2005 Oct 26.

A model of anthrax toxin lethal factor bound to protective antigen

D Borden Lacy  1 , Henry C Lin, Roman A Melnyk, Ora Schueler-Furman, Laura Reither, Kristina Cunningham, David Baker, R John Collier
Affiliations

A model of anthrax toxin lethal factor bound to protective antigen

D Borden Lacy et al. Proc Natl Acad Sci U S A. .

Abstract

Anthrax toxin is made up of three proteins: the edema factor (EF), lethal factor (LF) enzymes, and the multifunctional protective antigen (PA). Proteolytically activated PA heptamerizes, binds the EF/LF enzymes, and forms a pore that allows for EF/LF passage into host cells. Using directed mutagenesis, we identified three LF-PA contact points defined by a specific disulfide crosslink and two pairs of complementary charge-reversal mutations. These contact points were consistent with the lowest energy LF-PA complex found by using Rosetta protein-protein docking. These results illustrate how biochemical and computational methods can be combined to produce reliable models of large complexes. The model shows that EF and LF bind through a highly electrostatic interface, with their flexible N-terminal region positioned at the entrance of the heptameric PA pore and thus poised to initiate translocation in an N- to C-terminal direction.

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Figures

Fig. 1.
Fig. 1.
Mapping the PA- and LF-binding sites by site-directed mutagenesis. (a) The seven residues important for binding PA (D182, D187, L188, Y223, H229, L235, and Y236) cluster on the surface of LFN and are shown in green and red (15). (The D184A mutant did not show a binding defect but is depicted in pink, because it is likely to contribute to a net negative charge in this region.) Approximate dimensions of the site are indicated by the D187 Cα-H229 Cα distance (15 Å). The N terminus of the domain (E27) is shown in blue. (b) A surface rendering of the N-terminal domain 1′ (residues 175-258) from two PA63 subunits (yellow and pink) as viewed from the top of the heptameric ring. LFN binding studies to dimeric PA63 suggested two clusters of important residues shown in green and blue (8). One cluster contains residues P205, I207, I210, K214, K197, and R200 (subsite I). The second cluster contains R178 and the K197 and R200 residues from the neighboring PA63 subunit (subsite II). (Note: The P205A mutant was not tested in the dimeric background but is likely part of the first subsite because of its proximity to I207. K197 and R200, which were originally tested only in the context of subsite II, have recently been shown to participate in subsite I as well; H.C.L., unpublished work.) Approximate dimensions of the site are indicated by distances of the K214 Cα to the R200 Cα of the same subunit (16 Å) and the neighboring subunit (30 Å). Coordinates for LFN and the PA63-PA63 dimer were obtained from the 1J7N (12) and 1TZO (25) crystal structures, respectively. (c)LFN was manually docked on the PA dimer in two orientations that differed by ≈180°.
Fig. 2.
Fig. 2.
The Rosetta-Dock model of the LFN-PA dimer complex lies in a deep energy funnel. Three thousand independent trajectories were carried out, starting from the manually docked model. The energy for each structure (arbitrary units) is plotted against the rmsd between LFN molecules when the PA subunits from the manually docked and final models are aligned. There is a dramatic energy funnel around 20 Å from the starting model. The lowest energy structure also is the center of the largest cluster of low-energy models, and it is our most reliable model for this complex.
Fig. 3.
Fig. 3.
Identification of a disulfide crosslink between LFN Y108C and PA N209C. LFN Y108C and PA63 N209C form a disulfide-linked complex under oxidizing conditions (lane 1), which is disrupted in the presence of 10 mM DTT (lane 2).
Fig. 4.
Fig. 4.
A cell-surface binding assay shows that the LFN D187K-PA K213D/K213E and LFN E142K-PA K218E pairs can rescue binding defects. Data represent the fraction of mutant 35S-labeled LFN bound specifically to PA on cells relative to that of wild-type LFN. Error bars represent SEM.
Fig. 5.
Fig. 5.
The model of LFN bound to PA. (a) In this depiction of LFN (gray) bound to the surface of dimeric PA63 (light pink and yellow), the LFN E135, E142, and D187 residues are shown in red; the PA K197, K213 and K218 residues are shown in blue; and the LFN Y108 -PA N209 pair from the disulfide crosslinking experiment is shown in green. (Note: The E135 and K197 residues are predicted to interact based on the model but were not able to complement each other in a charge-reversal experiment.) As modeled, the bulk of LFN's contacts are with a single PA63 subunit (light pink), but contacts do exist with the neighboring PA63 subunit (yellow). The N- and C-terminal helices of LFN are shown in green and bright pink, respectively. (b) A close-up of the modeled interface between LFN and PA subsite I suggests a large number of electrostatic interactions and a buried LFN His residue. Residues from LFN and PA that may form electrostatic interactions are shown in red and blue, respectively. LFN H229 and Y236 were identified as the two most important residues for binding PA (15) and are shown in green, whereas the three important hydrophobic residues from the PA ligand-binding site (8) are shown in purple. (c) An aerial view of the PA heptamer-LFN complex in which three LFN molecules are bound. The footprint for LFN contacts on the PA dimer is shown in gray. LFN does not make contacts with residues R178 and R200 at subsite II. The three experimentally obtained PA contact points (K213, N209, and K218) are shown in red, green, and purple, respectively. The N- and C-terminal helices of LFN are shown in green and bright pink, respectively. (d) A cartoon side view of the LFN-PA complex in which only one LFN (blue) is bound. PA is depicted as a cartoon cutaway to emphasize the interior lumen of the heptameric ring. As modeled, the N-terminal helix of LFN (green) points toward the interior of the heptameric ring, whereas the C-terminal helix (bright pink) points upward and away from the heptamer, thereby allowing room for the LF catalytic domain. The N-terminal 26 residues of LFN are drawn in cartoon format as a black line and can potentially insert into the prepore lumen. LF translocation is thought to be initiated by the LF N terminus and occur through the lumen of the heptameric ring (11, 24).
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