Divalent metal ion coordination by residue T118 of anthrax toxin receptor 2 is not essential for protective antigen binding

Abstract

The protective antigen (PA) subunit of anthrax toxin interacts with the integrin-like I domains of either of two cellular receptors, ANTXR1 or ANTXR2. These I domains contain a metal ion-dependent adhesion site (MIDAS) made up of five non-consecutive amino acid residues that coordinate a divalent metal ion that is important for PA-binding. The MIDAS residues of integrin I domains shift depending upon whether the domain exists in a closed (ligand-unbound) or open (ligand-bound) conformation. Of relevance to this study, the MIDAS threonine residue coordinates the metal ion only in the open I domain conformation. Previously it was shown that the MIDAS threonine is essential for PA interaction with ANTXR1, a result consistent with the requirement that the I domain of that receptor adopts an open conformation for PA-binding. Here we have tested the requirement for the MIDAS threonine of ANTXR2 for PA-binding. We show that the toxin can bind to a mutant receptor lacking the MIDAS threonine and that it can use that mutant receptor to intoxicate cultured cells. These findings suggest that an open-like configuration of the ANTXR2 MIDAS is not essential for the interaction with PA.

Figure 1. ANTXR2 MIDAS metal ion bound by PA D683.

Close-up view of the PA-ANTXR2 generated with Molscript (PDB # ITZN) . The PA backbone is colored light blue with the D683 residue shown in cyan. The chelate Mg²⁺ ion is colored purple, the ANTXR2 Thr118 and Asp50 residues (mutated in this report) are shown in green with the other MIDAS residues represented in orange. Water molecules and oxygen atoms are represented by large and small red balls, respectively.

Figure 2. The cell surface T118A mutant ANTXR2 receptor binds PA.

(A) Flow cytometric analysis of PA binding to CHO-R1.1 cells and CHO-R1.1 cells engineered to express WT or mutant ANTXR2-EGFP fusion proteins. The cells were incubated with PA and then with a PA-specific antibody followed by an APC-labeled secondary antibody.

Figure 3. Soluble ANTXR2^T118A inhibits intoxication of cells.

WT and mutant sANTXR2 proteins were added in increasing amounts to measure their relative abilities to block CHO-K1 cell intoxication with PA (10⁻⁸ M) and LF_N-DTA (10⁻¹⁰ M). Cell viability was subsequently measured using the WST-1 assay and is represented as the percentage of that seen with cells incubated with LF_N-DTA alone (100% viable).

Figure 4. The cell surface T118A mutant ANTXR2 supports intoxication.

Triplicate samples of the cells shown in Fig. 2 were incubated with 10⁻¹⁰ M LF_N-DTA and varying concentrations of PA and cell viability was measured as described in the Fig. 3 legend.