doi:10.1016/j.jmb.2006.04.072 
Copyright © 2006 Published by Elsevier Ltd.
Self-association of the Transmembrane Domain of an Anthrax Toxin Receptor
Mandy Y. Go1, Sanguk Kim2, Anthony W. Partridge3, Roman A. Melnyk3, Arianna Rath3, Charles M. Deber3 and Jeremy Mogridge1, 
, 
1Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8
2Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang, Kyungbuk 790-784, South Korea
3Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8
Received 25 January 2006;
revised 25 April 2006;
accepted 29 April 2006.
Edited by G. von Heijne.
Available online 15 May 2006.
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Abstract
Protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted individually by Bacillus anthracis. These components of anthrax toxin must then assemble into complexes to intoxicate mammalian cells. Toxin assembly initiates when molecules of PA bind mammalian receptors ANTXR1/2 and are cleaved by surface proteases into 20 kDa and 63 kDa fragments. After PA20 dissociates, receptor-bound PA63 homo-oligomerizes into heptamers. Oligomeric PA63 binds EF and LF and these complexes are internalized into an acidic compartment where the two enzymatic components are translocated across the membrane by a channel formed by heptameric PA63. Since oligomerization of PA63 is required to bind and translocate the enzymatic components, we sought to determine whether interactions between toxin receptors could facilitate the assembly process. In the present work, we performed a co-immunoprecipitation experiment to demonstrate that ANTXR1 is oligomeric in mammalian cells. Computer modeling predicted the self-association of the ANTXR1 transmembrane domain and we detected oligomerization of ANTXR1 transmembrane domain peptides in the membrane-mimetic environment of SDS micelles using fluorescence resonance energy transfer. Furthermore, the ANTXR1 transmembrane domain mediated oligomerization of a reporter protein construct in a bacterial membrane. In both assays, mutations that disrupted the interaction were consistent with the interaction being mediated through an asymmetric binding interface. Mutations that impaired self-association of the transmembrane domain reduced the rate of PA63 heptamer formation on the mammalian cell surface. Our findings indicate that ANTXR1 transmembrane domains self-associate and that these interactions may stabilize intermediate oligomerization states of ANTXR1-PA63 complexes.
Keywords: anthrax; ANTXR1; transmembrane; fluorescence resonance energy transfer; TOXCAT
Abbreviations: PA, protective antigen; EF, edema factor; LF, lethal factor; ANTXR1, anthrax toxin receptor 1; ANTXR2, anthrax toxin receptor 2; TM, transmembrane; FRET, fluorescence resonance energy transfer; MBP, maltose-binding protein; CAT, chloramphenicol acetyl transferase; GpA, glycophorin A
Figure 1. Model of anthrax toxin assembly on the cell surface. PA binds to mammalian receptors and is cleaved by a cell-surface protease, releasing PA20. The remaining PA63 oligomerizes into heptamers, which bind a maximum of three EF/LF molecules. PA63 heptamerization causes receptors to cluster into lipid rafts and triggers receptor-mediated endocytosis into a low-pH endosome. The acidic environment triggers insertion of the heptamer into the membrane and translocation of EF/LF into the cytosol.
Figure 2. The ANTXR1 transmembrane domain. (a) The sequence of the ANTXR1 TM domain used for the modeling. (b) Helical wheel diagram of the ANTXR1 transmembrane helix. Residues in the a and d positions are implicated in mediating oligomerization in the symmetrical model, while residues in the c, d, f and g positions mediate the oligomerization of the asymmetrical model.
Figure 3. CD spectra of TM peptides. Representative spectra of peptides in the presence of 20 mM SDS. (a) ANTXR1-wt; (b) ANTXR1-a/d; (c) ANTXR1-b/e; and (d) ANTXR1-c/f.
Figure 4. FRET analysis of ANTXR1 TM peptides in SDS. (a) Representative fluorescence emission spectra (λexc = 341 nm) of dansyl-ANTXR1-wt in the absence (curve a) or presence (curve c) of dabsyl-ANTXR1-wt. Unlabeled ANTXR1-wt (curve b) or erythropoietin receptor TM domain (curve d) peptides were added to the mixture of dansyl-ANTXR1-wt and dabsyl-ANTXR1-wt. (b) Summary of FRET results measuring the percentage decrease in fluorescence intensity of 1 μM dansyl-labeled peptides in the presence of 1 μM dabsyl-labeled peptides (filled bars) and competed with 4 μM unlabeled peptides (open bars). (c) Reversal of quenching of 1 μM dansyl-ANTXR1-wt by 1 μM dabsyl-ANTXR1-wt in the presence of 4 μM unlabeled ANTXR1-wt, ANTXR1-a/d, ANTXR1-b/e or ANTXR1-c/f peptides. Error bars represent the standard error of the mean over three experiments for each peptide construct.
Figure 5. (a) malE complementation assay for correct topology of the TOXCAT chimerae. The constructs were transformed into malE-deficient E. coli NT326 and cultured on M9 agar containing 0.4% maltose and grown for two days at 37 °C. (b) CAT activity measurements of wild-type and mutant ANTXR1. The bars indicate the amount of CAT activity normalized to the GpA dimer. Both wild-type GpA and its disruptive mutant G83I are shown as controls. In addition, a construct containing a stop codon in the ToxR gene (ToxR-stop) is used as a negative control. Error bars represent the standard error of the mean of three measurements for each construct. *Significantly different from wild-type values (p < 0.05, Student's t-test).
Figure 6. ANTXR1 self-associates in mammalian cells. CHOR1.1 cells were transiently transfected with a plasmid expressing ANTXR1-HA (lanes 1 and 4) or ANTXR1-T7 (lanes 2 and 5) or were co-transfected with both plasmids (lanes 7 and 8) and then lysed. The lysate from cells expressing ANTXR1-HA was combined with the lysate from cells expressing ANTXR1-T7 (lane 6). Cell lysates were immunoprecipitated using anti-HA antibody (lanes 3–7) or control anti-GFP antibody (lane 8). Cell lysates (lanes 1 and 2) or immunoprecipitates (lanes 3–8) were immunoblotted with anti-T7 antibody. The immunoblot shown is representative of three independent experiments.
Figure 7. Mutations in the transmembrane domain of ANTXR1 reduce the rate of SDS-resistant [PA63]7 formation. (a) Representative Western blot of a toxin assembly assay. Wild-type and mutant receptor-expressing cells were treated with 10−8 M PA at 4 °C for 2 h, then shifted to 37 °C for the indicated times before lysis. Non-specific bands recognized by the anti-PA antibody are indicated by asterisks. Ratios of heptameric PA63 to monomeric PA83 and PA63 plotted against time for each of (b) ANTXR1-a/d, (c) ANTXR1-b/e, and (d) ANTXR1-c/f (each mutant represented by triangles) compared to wild-type (squares), averaged over three independent experiments. Error bars represent the standard error of the mean and slopes were compared using a Student's t-test.
Table 1.
Sequences of peptides derived from ANTXR1 TM (residues I318–W337)
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