X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism

Abstract

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.

Figure 1. Structure of the human CRT globular domain.

(A) Representation of the linear structure of CRT and of the construct used in this study. N, P, C, the discontinuous CRT segments as defined usually. Amino acid numbering is that of the unprocessed polypeptide. HAT, His-tag; fX cl., factor X cleavage site. (B) Two different views of the CRT globular domain structure. Regions from the N and C segments are colored light blue and dark blue, respectively. The Ca²⁺ ion is represented as a golden sphere. The approximate location of the P-domain insertion is indicated. (C) Structural alignment of human CRT and dog CNX. The positions of ß-strands and α-helices are indicated. The N-terminal extension and the linker are shown in pink. Grey italics show residues not defined in the structure. Residues aligned (−) or defining the common core of human CRT and rat CNX (+) are indicated. Residues included in the conserved clusters 1 and 2 are labeled 1 (green) and 2 (red), respectively.

Figure 2. Surface patches common to CRT and CNX.

(A) The more extended patch, cluster 1, corresponding to the identified lectin site . (B) The smaller cluster 2. CRT and CNX structures are in light blue and green, respectively. Labels correspond to CRT residues, and red labels indicate the most conserved ones. Hydrogen bonds are represented by dotted red lines. The two patches are displayed using stereograms.

Figure 3. The N-terminal extension of CRT interacts with the edge of the lectin site.

(A) Superposed views of the N-terminal extensions. (B) The interaction mode observed in the first crystal form. (C) The interaction mode observed in the second crystal form. (D) Superposition of the interacting regions of human CRT and the corresponding region of mouse CRT. Labels correspond to interacting residues of human CRT in (B) and (C), and to mouse CRT in (D). Hydrogen bonds and ionic interactions are represented by dotted red lines. The blue dotted lines in (A) represent disordered parts. F1, F2: crystal forms 1 and 2; m: mouse CRT. All items are displayed as stereograms.

Figure 4. The CRT molecular zipper.

(A) Schematic representation of the zipper. A red circle indicates the position of the binding site. Small circles locate the relative position of the SQDAR segment (pink) and its interacting residues in the neighboring molecule (yellow). The C-terminal helices are drawn in dark blue. (B) Negative-staining electron micrograph of the CRT construct. (C, D) Two orthogonal side- and top-views (the latter framed in A) of the interactions between CRT molecules defining the molecular zipper as seen in the crystal lattice. Residues involved in hydrogen and ionic bonds are shown in pink (segment SQDAR) and yellow. P marks the insertion position of the missing P domain. (E) Detailed view of the pocket lining the binding site as framed in (D). Exposed hydrophobic residues lining the pocket are in yellow; charged residues exposing a large hydrophobic surface (>65 Ų), forming a secondary shell, are in pale green; Residues shown by mutation to be essential for the chaperone function of CRT are in red (W319) and orange (H170).