Calnexin, More Than Just a Molecular Chaperone

Abstract

Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca²⁺-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.

Keywords: calcium binding protein; cell signaling; endoplasmic reticulum; molecular chaperone; protein–protein interactions.

Figure 1

Amino acid sequence alignment of calnexin from different species. The default Clustal X color scheme was used for similar/identical amino acid residues [42]: blue for hydrophobic, red for positively charged, magenta for negatively charged, green for polar, cyan for aromatic, pink for cysteine, orange for glycine, yellow for proline and no color for amino acid residues that are not conserved. Dashes represent gaps in the amino acid sequence. The N-terminal signal sequence, intraluminal domain, transmembrane domain and C-terminal domain are indicated based on human calnexin protein topology. The numbering of amino acid residues referred to in the figure as well as the text corresponds to the mature human calnexin protein. Alignment was performed using Multiple Alignment using Fast Fourier Transform (MAFFT) high speed multiple sequence alignment program (https://toolkit.tuebingen.mpg.de/tools/mafft; accessed 31 August 2022). Jalview [43] was used to visualize amino acid alignment. Additional calnexin sequences from different species are shown in Supplementary Figure S1.

Figure 2

Schematic linear sequence of the calnexin gene and its encoded protein. The human calnexin gene is located on a forward strand of distal end of the long arm of chromosome 5 and is comprised of 15 exons (of which 14 are protein-coding exons) transcribed to a mature transcript of 4915 bp that is translated into a 592 amino acid residue polypeptide. Linear schematic representation of the calnexin protein and the corresponding exons encoding the specific protein domains of calnexin. The white boxes in the gene schematic diagram correspond to the untranslated regions of the first and last exons. The calnexin protein schematic diagram shows the signal peptide (red), the luminal domain (green for the N-domain and blue for the P-domain), the transmembrane domain (TM, yellow) and the cytosolic C-terminal domain (orange). The hatched box represents a portion of the ER membrane. Four repeats of Motif 1 and four repeats of Motif 2 are labelled as “11112222” and depict the proline-rich amino acid sequence repeats in the P-domain. The ER retention signal (RKPRRE) is shown as the most distal C-terminal amino acid sequence.

Figure 3

Schematic representation of full-length membrane-embedded calnexin. Calnexin (Protein Data Bank DOI: 10.2210/pdb1JHN/pdb) has three domains: luminal domain (comprised of N and P subdomains), transmembrane domain and C-terminal domain (no experimental structural information available). The yellow circle depicts a bound Ca²⁺ ion. The numbering of amino acid residues in the C-terminal domain is relative to the mature N-terminus. The transmembrane domain was modelled based on molecular dynamics simulation performed by Lakkaraju et al. [34] and shows that Pro⁴⁹⁴ introduces a kink in the helix located approximately at the midpoint of the domain. The C-terminal domain was modelled using AlphaFold [98]. The calnexin C-terminal domain undergoes distinct post-translational modifications including palmitoylation at Cys⁴⁸² and Cys⁴⁸³ [35] (shown in blue); sumoylation at Lys⁵⁰⁵ [96] (shown in green); and phosphorylation at Ser⁵³⁴, Ser⁵⁴⁴ and Ser⁵⁶³ [97] (shown in red); Asp⁵³⁹ proteolytic cleavage site (shown in black). Known and potential sites of post-translational modifications in the calnexin C-terminal domains of various species are shown in Supplementary Figure S1.

Figure 4

Crystal structure of the calnexin luminal domain and its characteristics. (A) Crystal structure of the calnexin intraluminal domain. The globular N-globular domain is shown in green while the P-domain is depicted in yellow. (B) Calnexin putative Ca²⁺-binding site, showing a Ca²⁺ ion (yellow circle) coordinated by Asp⁴¹⁶, Asp⁹⁷, Ser⁵⁴ and potentially Lys⁹⁸. (C) Calnexin carbohydrate binding site, showing the sidechains of Tyr¹⁴⁴, Lys¹⁴⁶, Tyr¹⁶⁵, Glu¹⁹⁶, Glu⁴⁰⁵ and Met¹⁶⁸ involved in the binding of carbohydrate moieties.