Pekka Määttänen

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.

A structural overview of the PDI family of proteins.

Protein disulfide isomerases (PDIs) are enzymes that mediate oxidative protein folding in the endoplasmic reticulum. Understanding of PDIs has historically been hampered by lack of structural information. Over the last several years, partial and full‐length PDI structures have been solved at an increasing rate. Analysis of the structures reveals common features shared by several of the best known PDI family members, and also unique features related to substrate and partner binding sites. These exciting breakthroughs provide a deeper understanding of the mechanisms of oxidative protein folding in cells.

Solution structure of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin‐like domains (abb′a′). Cross‐linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b′. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b′, respectively) by triple‐resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b′ domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase‐catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb′ fragment. Our findings provide a structural basis for previous kinetic and cross‐linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate‐binding site.

Structural Basis of Cyclophilin B Binding by the Calnexin/Calreticulin P-domain.

Little is known about how chaperones in the endoplasmic reticulum are organized into complexes to assist in the proper folding of secreted proteins. One notable exception is the complex of ERp57 and calnexin that functions as part the calnexin cycle to direct disulfide bond formation in N-glycoproteins. Here, we report three new complexes composed of the peptidyl prolyl cis-trans-isomerase cyclophilin B and any of the lectin chaperones: calnexin, calreticulin, or calmegin. The 1.7 Å crystal structure of cyclophilin with the proline-rich P-domain of calmegin reveals that binding is mediated by the same surface that binds ERp57. We used NMR titrations and mutagenesis to measure low micromolar binding of cyclophilin to all three lectin chaperones and identify essential interfacial residues. The immunosuppressant cyclosporin A did not affect complex formation, confirming the functional independence of the P-domain binding and proline isomerization sites of cyclophilin. Our results reveal the P-domain functions as a unique protein-protein interaction domain and implicate a peptidyl prolyl isomerase as a new element in the calnexin cycle.

An Interaction Map of Endoplasmic Reticulum Chaperones and Foldases

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.

ERp57 does not require interactions with calnexin and calreticulin to promote assembly of class I histocompatibility molecules and it enhances peptide loading independently of its redox activity

ERp57 is a thiol oxidoreductase that catalyzes disulfide formation in heavy chains of class I histocompatibility molecules. It also forms a mixed disulfide with tapasin within the class I peptide loading complex, stabilizing the complex and promoting efficient binding of peptides to class I molecules. Since ERp57 associates with the lectin chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, we examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells. Moreover, ERp57 formed a mixed disulfide with tapasin and promoted efficient peptide loading in the absence of interactions with calnexin and calreticulin. These findings suggest that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin chaperones. We also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide binding site in substrate recognition. Finally, by inactivating the redox-active sites of ERp57, we demonstrate that its enzymatic activity is dispensable in stabilizing the peptide loading complex and in supporting efficient peptide loading. Thus, ERp57 appears to play a structural rather than catalytic role within the peptide loading complex.

Structure of the noncatalytic domains and global fold of the protein disulfide isomerase ERp72.

Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

Structure of the Catalytic a(0)a Fragment of the Protein Disulfide Isomerase ERp72.

Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). The ER contains many different PDI-like proteins. Some, such as PDI, are general enzymes that directly recognize misfolded proteins while others, such as ERp57 and ERp72, have more specialized roles. Here, we report the high-resolution X-ray crystal structure of the N-terminal portion of ERp72 (also known as CaBP2 or PDI A4), which contains two a(0)a catalytic thioredoxin-like domains. The structure shows that the a(0) domain contains an additional N-terminal beta-strand and a different conformation of the beta5-alpha4 loop relative to other thioredoxin-like domains. The structure of the a domain reveals that a conserved arginine residue inserts into the hydrophobic core and makes a salt bridge with a conserved glutamate residue in the vicinity of the catalytic site. A structural model of full-length ERp72 shows that all three catalytic sites roughly face each other and positions the adjacent hydrophobic patches that are likely involved in protein substrate binding.

1H, 13C and 15N resonance assignments of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase (PDI) participates in protein folding and catalyses formation of disulfide bonds. The b′ domain of human PDI contributes to binding unfolded proteins; its structure is stabilized by the b domain. Here, we report NMR chemical shift assignments for the bb′ fragment.

Chaperones in the Endoplasmic Reticulum (ER): Function and Interaction Network

The directional entry, oxidative folding, and quality control of proteins that enter the secretory pathway is mediated by chaperones and foldases in and adjacent to the endoplasmic reticulum (ER). Properly folded and assembled proteins continue along the secretory pathway while proteins that ultimately fail quality control are targeted to the proteasome by removal from the ER in a process called ER-associated degradation (ERAD). The protein folding machineries in the ER interact with each other to form functional complexes. Studies have revealed that abundant chaperones and foldases serve multiple functions in the ER through membership in diverse complexes that can target their activities to substrates at different stages of maturation. These findings are providing insight into how ER complexes combine various functions together to engage substrates and determine their fates.