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Review
. 2020 Oct;287(20):4322-4340.
doi: 10.1111/febs.15330. Epub 2020 Apr 27.

Calnexin cycle - structural features of the ER chaperone system

Guennadi Kozlov  1 , Kalle Gehring  1
Affiliations
Review

Calnexin cycle - structural features of the ER chaperone system

Guennadi Kozlov et al. FEBS J. 2020 Oct.

Abstract

The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N-glycosylated proteins. Recent structures of components of the calnexin cycle have deepened our understanding of quality control mechanisms and protein folding pathways in the ER. In the calnexin cycle, proteins carrying monoglucosylated glycans bind to the lectin chaperones calnexin and calreticulin, which recruit a variety of function-specific chaperones to mediate protein disulfide formation, proline isomerization, and general protein folding. Upon trimming by glucosidase II, the glycan without an inner glucose residue is no longer able to bind to the lectin chaperones. For proteins that have not yet folded properly, the enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by adding a glucose back to the N-glycan. This allows the misfolded proteins to re-associate with calnexin and calreticulin for additional rounds of chaperone-mediated refolding and prevents them from exiting the ERs. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function-specific chaperones and how UGGT recognizes misfolded proteins.

Keywords: CypB; ERp29; ERp57; PDI; UGGT; calnexin; calnexin cycle; calreticulin; endoplasmic reticulum; protein folding.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Overview of calnexin/calreticulin cycle. (A) The monoglucosylated form of newly synthesized glycoproteins proteins binds to calreticulin (CRT)/calnexin (CNX) and promotes protein folding with assistance from ERp57, CypB, and ERp29. Following release of the terminal glucose by glucosidase II, natively folded proteins are transported to Golgi. Incompletely folded proteins are reglucosylated by UGGT and rebind calreticulin/calnexin for additional folding cycles. If multiple folding cycles are unsuccessful, terminally misfolded proteins are transported to the cytoplasm for degradation via the ER‐associated protein degradation (ERAD) pathway. (B) Structure of N‐linked glycan. The precursor glycan is attached to the protein with three glucose residues. The first two are removed through the action of glucosidases I and II to generate the monoglucosylated form that is required for binding calnexin and calreticulin. UGGT acts on misfolded glycoproteins to add back glucose to the glycan for additional rounds of chaperone‐mediated folding.
Fig. 2
Fig. 2
Calnexin/calreticulin structure. (A) Domain architecture of calnexin (CNX), calmegin (CMG), calreticulin (CRT), and calreticulin 3 (CRT3). The P‐domain is inserted into the lectin domain, while calnexin and calmegin also possess a transmembrane (TM) domain. The P‐domain in calnexin and calmegin is composed of four repeated modules, while the domain in calreticulin and calreticulin 3 contains only three modules. (B) Overlay of calnexin (PDB 1JHN) and calreticulin (PDB 3RG0 and 6ENY) structures illustrates flexibility of the P‐domain and the site of the glycan (red/white) bound to the lectin domain. In the peptide‐loading complex (PDB 6ENY), the C terminus of calreticulin forms a long helix, but it is unlikely to be folded in solution. The C termini of both proteins are rich in acidic residues that bind Ca2+ ions. (C) Four sugars of the glycoprotein glycan bind to the lectin domain along the β‐sheet surface (PDB 3O0W). The sugar‐binding specificity arises from the numerous hydrogen bonds between the glycan and protein. A disulfide bridge between Cys105 and Cys137 interacts with the Man(4) moiety. (D) High‐affinity Ca2+‐binding site in the lectin domain of calreticulin (PDB 3O0W). (E) Each repeated module in the P‐domain contains a small hydrophobic core of two tryptophans and a lysine residue. Residues from the calreticulin P‐domain structure (PDB 5V90) show the close packing of the hydrophobic van der Waals surfaces.
Fig. 3
Fig. 3
Interactions of calnexin/calreticulin with other ER proteins. (A) Domain architecture of calnexin/calreticulin‐binding partners. The asterisks mark domains required for interactions with calnexin/calreticulin. The tip of P‐domain (cyan) interacts with very different structural scaffolds from protein disulfide isomerase ERp57 (B), cis‐trans prolyl isomerase CypB (C), and general chaperone ERp29 (D). Met257 and Asp258 are part of the conserved Met‐Asp‐Gly sequence at the tip of P‐domains instrumental for the binding. Positively charged residues Arg282 (ERp57), Lys97 (CypB), and Arg223 (ERp29) indispensable for interactions with calnexin/calreticulin are also shown as sticks. Crystal structure of calreticulin P‐domain (PDB 5V90) was overlaid with cryo‐EM structure of P‐domain bound to ERp57 (PDB 6ENY) and with a shorter segment of P‐domain bound to CypB (PDB 3ICI). The modeled parts of P‐domains are shown with dashed lines. The ERp57‐P‐domain model was assembled using the high‐resolution structure of ERp57 bb′ domain fragment (PDB 2H8L) and relative orientation of both proteins in cryo‐EM structure.
Fig. 4
Fig. 4
Structure of UDP‐glucose:glycoprotein glucosyltransferase. (A) Domain architecture of UGGT. The Sep15‐binding site is marked with an asterisk. (B) Cartoon representation of UGGT full‐length structure from Chaetomium thermophilum (PDB 5MZO) shows four DsbA‐like domains TRXL1 (blue), TRXL2 (cyan), TRXL3 (green), and TRXL4 (yellow) followed by β‐rich domain (pink) and catalytic domain (gray). Location of UDP‐glucose is modeled from the structure of the Thermomyces dupontii UGGT catalytic domain with bound UDP‐glucose (PDB 5H18). DsbA‐like domains of UGGT (C) have stronger resemblance to bacterial oxidoreductase DsbA (PDB 5KBC; magenta/gray) (D) than to thioredoxin‐like domains in mammalian PDI (PDB 3F8U; orange). Surprisingly, the helical bundle (purple) is at the N terminus of TRXL1 domain, while the TRXL4 domain is assembled from nonconsecutive stretches (yellow and brown) of the primary sequence. The C‐terminal domain of Sep15 (PDB 2A4H; dark gray) represents a minimalist version of a redox fold. (E) The UGGT catalytic domain is similar to other glycosyltransferases. The structure of UGGT catalytic domain (PDB 5H18) with bound UDP‐glucose (cyan) suggests that the acceptor glycan in UGGT binds in a surface cavity formed by a distorted helix next to the glucose moiety of UDP‐glucose. In the structure of the GT8 glycosyltransferases LgtC (PDB 1GA8), this pocket is occupied by the acceptor lactose (purple). Two aspartates in the UGGT catalytic site coordinate a calcium ion. (F) Interdomain mobility of UGGT. Comparison of the open (PDB 5MZO) and closed (PDB 5N2J) UGGT structures of Ch. thermophilum UGGT shows significant differences in positions of the TRXL2 and TRXL3 domains. The catalytic domain has been omitted to better show the conformational changes.
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