Structural Basis of Carbohydrate Recognition by Calreticulin

Abstract

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc 1 Man 9 GlcNAc 2 glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide ␣-Glc-

Calreticulin Is a Thermostable Protein with Distinct Structural Responses to Different Divalent Cation Environments

“…Asp 311 within the globular domain is a key residue that defines the high affinity calcium-binding site (Table 1). These results confirm findings of the nature of the high affinity calcium-binding site of calreticulin from the crystal structure of its globular domain (4). Additionally, the studies reveal the presence of low affinity calcium-binding sites within the C terminus of the globular domain and the N terminus of the acidic domain that appear to be independent of the rest of the acidic domain.…”

“…2A and Table 1) indeed corresponds to the occupancy of a high affinity site that is not located in the acidic C terminus of calreticulin or its P-domain. Furthermore, mCRT(D311A) displayed an inability to bind calcium at the high affinity site (Table 1), a finding consistent with the calcium-binding site identified from the crystal structures of the calreticulin globular domain (PDB codes 3O0V, 3O0W, and 3O0X) and of soluble calnexin (PDB code 1JHN) (3,4) (Fig. 1A).…”

“…This destabilizing conformational change may occur via disruption of electrostatic interactions between the long C-terminal helix in the globular domain and the rest of the calreticulin molecule. Analysis of the crystal structure of the globular domain of calreticulin (PDB code 3O0V) (4) Previous findings indicated that the isolated acidic domain of calreticulin is able to fold and attain a more compact form as it binds calcium (16). However, although it could be the case that the acidic domain of calreticulin is folded independently of its globular domain in high calcium environments (Fig.…”

“…In calreticulin, the presence of a high affinity calcium-binding site within the P-domain has been suggested based on biochemical studies of 45 Ca 2ϩ binding to calreticulin truncation constructs (1,7). However, based on crystallographic studies of calreticulin and calnexin, Asp 311 and other residues within the globular domain of calreticulin are predicted to be involved in high affinity calcium binding (3,4) (Fig. 1, A and C).…”

“…The recently reported crystal structures of the globular domain of calreticulin (PDB codes 3O0V, 3O0W, and 3O0X) have revealed a high degree of conservation with calnexin in the overall structure and in the residues corresponding to the high affinity calcium (Fig. 1A) and glycan-binding sites (4). Calnexin and calreticulin also contain a proline-rich domain, the P-domain (Fig.…”