Structural basis of carbohydrate recognition by calreticulin

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 Glc₁Man₉GlcNAc₂ 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 α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave β-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O₂ of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly¹²⁴ and the side chain of Lys¹¹¹. The structure also explains a requirement for the Cys¹⁰⁵–Cys¹³⁷ disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys¹⁰⁵–Cys¹³⁷ disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc₁Man₃ tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.