The structure of tumor endothelial marker 8 (TEM8) extracellular domain and implications for its receptor function for recognizing anthrax toxin

Abstract

Anthrax toxin, which is released from the gram-positive bacterium Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 A resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA (consisting of residues 56, 57, and 154-160) is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.

Figure 1. TEM8 vWA domain hexamer in the crystal cell.

Protein molecules are shown in ribbon presentation. Six TEM8 vWA domain molecules (labeled A–F) form two trimers, which further assemble into a ball shape in the asymmetric unit. Each TEM8 molecule is shaded with a unique color. The dimensions of the ball are ∼82×82×66 Å.

Figure 2. Overall structure of the TEM8 vWA domain and its open conformation.

(A) TEM8 vWA domain structure (side view). Five parallel β strands (β₁, β_2, β₄, β₅, and β₆) and one short anti-parallel β-strand (β₃) form a central sheet that is surrounded by six α-helices (α₁–α₆). This structure contains a chelated Mg²⁺ ion (light green sphere) in the MIDAS site, with a bound pseudo-ligand contributed by an acetate ion (ACT1, purple). Two additional acetate ions (ACT2 and ACT3, purple) are also observed in the structure. (B) TEM8 MIDAS site (top view). Ser52 and Ser54 in helix α1 (blue) and Thr118 in α3 (green) form direct bonds that coordinate the Mg²⁺ ion, while Asp50 in α1 (blue) and Glu152 and Asp150 in the α4–β4 loop (yellow) form water-mediated hydrogen-bonds to the metal ion. The rest of the MIDAS coordination site is occupied by a mimic ligand (i.e., the acetate ion, ACT1, purple). (C) TEM8 vWA domain structure (colored clay) was superimposed onto the CMG2-S38 vWA domain structure (colored green, PDB ID 1SHU). The most distinct sites are highlighted by pink ovals. (D) The superposition (top view) between the TEM8 vWA domain structure (clay), domain I structure of Integrin CR3 in the open conformation (purple, 1IDO) and CR3 in the closed conformation (blue, 1JLM). The TEM8 vWA structure is much closer to the open conformation.

Figure 3. TEM8-PA₆₃ complex model and details of their interaction.

(A) A model of PA₆₃ and TEM8 vWA domain complex (PA₆₃, cyan; TEM8 vWA domain, yellow; Mg²⁺, green; and acetate ion, purple). The TEM8 vWA domain is superimposed onto the CMG2 vWA domain according to the structure of the CMG2-PA complex (PDB ID 1T6B). (B–E) Comparison of the TEM8-PA₆₃ binding surface and CMG2-PA₆₃ binding surface. According to our TEM8-PA₆₃ complex model, there are four TEM8 regions anticipated to interact with PA₆₃. Here, we show detailed structural differences between the TEM8-PA₆₃ binding surface and the CMG2-PA₆₃ binding surface (TEM8, yellow; CMG2, purple; and PA₆₃, cyan). The molecular surface of PA (cyan, semi transparent) is also included. (B) Part 4 (residues 153–158), located in the β3–β4 loop interacts, with Leu340 and Ala341 in domain 4 of PA₆₃. (C) Part 3 (residues 113 and 115), located in the α2–α3 loop, interacts with a hydrophobic cleft comprised of Leu687, Ile689, Ile646, Phe678, and Ile656 of PA₆₃. The picture is reverse with box in A (D) Part 2 (residues 87 and 88), located in the β2–β3 loop, interacts with Asp657, Arg658, Asp714, and Thr715 of PA₆₃. (E) Part 1 (residues 56 and 57), located in helix α1, interacts with Tyr688 in domain 2 of PA₆₃.

Figure 4. Inhibition ability of receptor variants (vWA domain) for protecting J774A-1 cells from PA intoxication.

The survival rate is calculated using the equation:Control 1 is cells treated without either PA or receptors (mutants) and control 2 is cells treated with PA but without receptors. (A) The protective ability (IC₅₀) of TEM8 variants that replace the original residue with Ala or contrary charged residue at the conserved sites in the binding interface between TEM8 and CMG2. (B) The protective ability (IC₅₀) of TEM8 variants that replace the original residue with the corresponding residue in CMG2 at the non-conserved sites. Data points and error bar represent the mean ± SEM values for three independent experiments in (A) and (B). (C) Survive curves show the negative Log value of the IC₅₀ by TEM8 variants/receptors, based on results showed by (A). In the same way, (D) is the corresponding curves of (B). Data points and error bar represent the mean ± SEM values for one representative experiment with duplicates in (C) and (D).

Figure 5. Comparison of PA-CMG2 and PA M662R/R659S-CMG2.

(A) PA-CMG2 complex (light blue, PDB ID 1T6B). The TEM8 vWA domain, colored yellow, is superimposed onto the CMG2 vWA domain of the complex. (B) Model of the PA mutant (M662R/R659S) complexed with CMG2, according to the structure of the PA-CMG2 complex. The TEM8 vWA domain was superimposed onto the CMG2 vWA domain. The mutated residue is colored grey.

Figure 6. pH threshold of pore formation of TEM8 variants.

Formation of SDS-PAGE resistant oligomers is assayed at different pH values in solution with the vWA domain of the WT receptors and TEM8 mutants. Experiments have been repeated at least twice and the results were identical.