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 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 O(2) of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly(124) and the side chain of Lys(111). The structure also explains a requirement for the Cys(105)-Cys(137) disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys(105)-Cys(137) disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc(1)Man(3) 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.
Highlights
Protein glycosylation plays an important role in maturation and quality control of proteins in the endoplasmic reticulum (ER)3 [1,2,3]
Crystallization and Structure Determination of CRT Lectin Domain—Previous attempts to crystallize CRT were likely hindered by the intrinsic mobility of the arm-like P-domain and unstructured C terminus
Characterization by mass spectrometry of fragments from trypsin digestion suggested the C terminus could be removed by cleavage at Lys368 to produce a stable fragment and NMR spectroscopy confirmed that it was soluble and well folded
Summary
Protein glycosylation plays an important role in maturation and quality control of proteins in the endoplasmic reticulum (ER)3 [1,2,3]. The glucose and adjacent three mannose residues are critically important for carbohydrate recognition by CNX and CRT (Scheme 1) (4 – 6). Removal of the terminal glucose from correctly folded protein substrates releases them from binding to CNX or CRT and allows their exit from the ER to their final destination. The lectin domain shows a fold similar to leguminous lectins and largely consists of a -sandwich formed by two curved -sheets. It contains a single high affinity calcium-binding site that plays an important role in stabilizing the protein [9] but does not participate in carbohydrate recognition [8].
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