Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor

Abstract

Anthrax toxin is released from Bacillus anthracis as three monomeric proteins, which assemble into toxic complexes at the surface of receptor-bearing host cells. One of the proteins, protective antigen (PA), binds to receptors and orchestrates the delivery of the other two (the lethal and edema factors) into the cytosol. PA has been shown to bind to two cellular receptors: anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (CMG2). Both are type 1 membrane proteins that include an approximately 200-aa extracellular von Willebrand factor A (VWA) domain with a metal ion-dependent adhesion site (MIDAS) motif. The anthrax toxin receptor/tumor endothelial marker 8 and CMG2 VWA domains share approximately 60% amino acid identity and bind PA directly in a metal-dependent manner. Here, we report the crystal structure of the CMG2 VWA domain, with and without its intramolecular disulfide bond, to 1.5 and 1.8 A, respectively. Both structures contain a carboxylate ligand-mimetic bound at the MIDAS and appear as open conformations when compared with the VWA domains from alpha-integrins. The CMG2 structures provide a template to begin probing the high-affinity CMG2-PA interaction (200 pM) and may facilitate understanding of toxin assembly/internalization and the development of new anthrax treatments. The structural data also allow molecular interpretation of known CMG2 VWA domain mutations linked to the genetic disorders, juvenile hyaline fibromatosis, and infantile systemic hyalinosis.

Fig. 1.

Structure of the CMG2 VWA domain. A ribbon diagram of the S38 structure indicates secondary structure elements. Highlighted amino acid residues include the N- and C-terminal cysteines (C39 and C218, respectively) that form a disulfide bond (the sulfur atoms are depicted in yellow) and the conserved amino acids of the MIDAS motif. The Mg²⁺ ion is shown as a large blue sphere with two bound water molecules depicted as beige spheres. The small red spheres correspond to oxygen atoms within the MIDAS amino acids. The E194 residue from a neighboring CMG2 molecule (only E194 is shown in pink) contributes the sixth coordinating residue at the MIDAS metal. The structures of S38 and R40 superimpose with an rms deviation of 0.7 Ų. They differ primarily in the orientation of the α6 C-terminal helix. This helix and its preceding loop in the R40 structure are depicted in blue. This image and Fig. 3 were generated by the program molscript (47) and rendered in raster3d (48).

Fig. 2.

Sequence alignment of the VWA domains from CMG2, ATR/TEM8, and the αM integrin. The sequences were aligned with clustalw (49) and were displayed in espript (50) along with the secondary structure assignments for CMG2 (Top) and the open conformation of the αM integrin (PDB ID code 1IDO, Bottom; ref. 23). η,3₁₀-helix. White lettering boxed with a red background indicates residues that are conserved in all three sequences, and red lettering indicates similar residues. The numbering (Top) corresponds only to the CMG2 sequence.

Fig. 3.

The CMG2 VWA domain is in an open conformation. (a) The backbone structure of the CMG2 VWA domain (light green) was superimposed onto the aligned structures of the αM integrin I domains in their open (dark green, PDB ID code 1IDO) and closed (blue, PDB ID code 1JLM) conformations (23, 26). The hydrophobic pockets I and II are indicated by gray ovals and the Mg²⁺ ion for CMG2 is depicted as a blue sphere. (b) The closed conformation of αM with Phe-302 buried in hydrophobic pocket I and Ile-316 buried in hydrophobic pocket II. (c) The open conformation of αM shows a shift in the C-terminal helix from its position in b such that Phe-302 becomes solvent-exposed, and hydrophobic pocket II is now occupied by Leu-312. The positions of Phe-275 and Gly-243 have also shifted. (d) The structure of the CMG2 VWA domain is similar to that of c and is therefore an open conformation. It is hypothesized that the presence of Ile-213 bound in hydrophobic pocket II and the absence of a downstream hydrophobic residue equivalent to αM Ile-316 might help stabilize the open conformation. Residues that, when mutated, result in ISH and JHF disease (Leu-45, Gly-105, Ile-189, and Cys-218) are depicted in yellow. (e) In the closed structure of αM, the Mn²⁺ ion (blue sphere) is coordinated by three waters, two MIDAS serines, and an aspartic acid. The bond to the MIDAS threonine has been broken.(f) In the open structure of αM, the Mg²⁺ ion is coordinated directly by two serines, two waters (medium red spheres), a threonine, and a glutamate from a neighboring monomer. (g) The coordination of the MIDAS metal in the CMG2 VWA domain structure is identical to the coordination observed for the open conformation of αM shown in f.