Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Abstract

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K d for peptides of 0.5-1 M. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function. Soluble calreticulin (Crt)7 and membrane-bound calnexin (Cnx) are glycoprotein-specific chaperones of the endoplasmic reticulum (ER). They are components of the quality control machinery that prevents the premature release of folding intermediates from the ER, and they promote proper folding by preventing aggregation and by providing a suitable environment in which the associated ERp57 enzyme catalyzes thiol oxidation and isomerization. If proper folding cannot be achieved, the glycoproteins are marked for ER-associated degradation (1-4).Crt and the ER luminal portion of Cnx share a similar structure (5-7). They consist of a globular lectin domain and an elongated hairpin-like arm domain, the tip of which comprises the binding site for ERp57 (8,9). The arm domain is also known as the P domain because it consists of two proline-rich sequence motifs that are repeated in tandem three or four times in Crt and Cnx, respectively. Whereas the structure of the ER luminal portion of Cnx has been solved (7), only the individual lectin and arm domain structures have been determined for Crt by x-ray (6) and NMR (5) methods, respectively. These studies revealed the presence of a bound Ca 2ϩ ion within the lectin domain that is crucial for chaperone stability (10, 11) as well as the location of the oligosaccharide binding site. The lectin domain of both chaperones binds monoglucosylated N-linked oligosaccharides on newly synthesized glycoproteins (12-14), and the removal and re-addition of the terminal glucose residue on these oligosaccharides regulates lectin-based cycles of chaperone interaction (15). ...

Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells

“…The ability of these chaperones to suppress client protein aggregation appears to be due in part to their sequestration of glycoprotein folding intermediates between the globular lectin domain and the flexible extended arm domain. This is suggested on the basis of FRET experiments using probes within the two domains (22) as well as arm domain truncation mutants that exhibited reduced abilities to suppress aggregation in vitro (23,24).…”

“…Substrates-Previously, we showed that Crt suppresses the aggregation of non-glycosylated client proteins through a polypeptide binding site that resides within its globular lectin domain but at a site distinct from the site of oligosaccharide binding (24). To localize the polypeptide binding site more precisely, we turned to our crystal structure of the mouse Crt lectin domain (4) and identified four surface-exposed hydrophobic patches at locations removed from the lectin site that could potentially mediate aggregation suppression (Fig.…”

“…The ability of these chaperones to suppress client protein aggregation appears to be due in part to their sequestration of glycoprotein folding intermediates between the globular lectin domain and the flexible extended arm domain. This is suggested on the basis of FRET experiments using probes within the two domains (22) as well as arm domain truncation mutants that exhibited reduced abilities to suppress aggregation in vitro (23,24).There is also substantial evidence supporting the existence of polypeptide-based interactions between Cnx or Crt and their client proteins, interactions that may also contribute to the suppression of aggregation. This is based on the finding that binding of these chaperones to a number of clients is unaltered or only modestly diminished in glucosidase-deficient cells, cells …”

“…Furthermore, both Cnx and Crt bind non-glycosylated hydrophobic peptides in vitro (23,24,26) and are capable of suppressing the in vitro aggregation of non-glycosylated proteins under physiological conditions of the ER (24,27,28). This aggregation suppression function resides at a single site within the globular domain and can be blocked by the addition of hydrophobic peptides (23,24). Efforts have been ongoing to localize more precisely the site responsible for aggregation suppression with most emphasis placed on Crt.…”

“…Efforts have been ongoing to localize more precisely the site responsible for aggregation suppression with most emphasis placed on Crt. Several protein binding sites have been identified on Crt such as those specific for GABARAP (29) and thrombospondin (30), but interacting peptides derived from these proteins fail to compete in aggregation suppression assays, indicative of distinct binding sites (24). Additional binding sites have been proposed in proximity to the lectin site (22,31,32), but these either do not exhibit the expected specificity for hydrophobic substrates (22) or would be expected to be blocked upon addition of monoglucosylated oligosaccharide (31), a treatment that does not affect the binding of hydrophobic peptides to Crt (24).…”