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 ␣-Glc-

Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltagedependent general diffusion porin. The transmembrane domain of FomA forms a b-barrel that is predicted to consist of 14 b-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded b-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a b-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.

The outer-membrane proteins OmpA and FhuA of Escherichia coli are monomeric beta-barrels of widely differing size. Polarized attenuated total reflection infrared spectroscopy has been used to determine the orientation of the beta-barrels in phosphatidylcholine host matrices of different lipid chain lengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples are used to deduce the strand tilt (beta = 46 degrees for OmpA and beta = 44.5 degrees for FhuA). These values are then used to deduce the order parameters, P(2)(cos alpha), of the beta-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the beta-barrels and their assembly in the membrane are discussed in terms of hydrophobic matching with the lipid chains.

The stability of OmpA in large unilamellar vesicles of dilauroyl phosphatidylcholine was studied using different concentrations of urea. The effective energy of unfolding, as determined from refolding experiments, is greater than that for small sonicated unilamellar vesicles by an amount that is compatible with estimates of the elastic energy of highly curved vesicles. The on-rate for refolding and insertion is slower for large unilamellar vesicles than for small unilamellar vesicles, which indicates a contribution of vesicle strain also to the free energy of the transition state.

Amphipols are a class of amphipathic polymers designed to maintain membrane proteins in aqueous solutions in the absence of detergents. Denatured β-barrel membrane proteins, like outer membrane proteins OmpA from Escherichia coli and FomA from Fusobacterium nucleatum, can be folded by dilution of the denaturant urea in the presence of amphipol A8-35. Here, the folding kinetics and stability of OmpA in A8-35 have been investigated. Folding is well described by two parallel first-order processes, whose half-times, ~5 and ~70 min, respectively, are independent of A8-35 concentration. The faster process contributed ~55-64 % to OmpA folding. Folding into A8-35 was faster than into dioleoylphosphatidylcholine bilayers and complete at ratios as low as ~0.17 g/g A8-35/OmpA, corresponding to ~1-2 A8-35 molecules per OmpA. Activation energies were determined from the temperature dependence of folding kinetics, monitored both by electrophoresis, which reports on the formation of stable OmpA tertiary structure, and by fluorescence spectroscopy, which reflects changes in the environment of tryptophan side chains. The two methods yielded consistent estimates, namely ~5-9 kJ/mol for the fast process and ~29-37 kJ/mol for the slow one, which is lower than is observed for OmpA folding into dioleoylphosphatidylcholine bilayers. Folding and unfolding titrations with urea demonstrated that OmpA folding into A8-35 is reversible and that amphipol-refolded OmpA is thermodynamically stable at room temperature. Comparison of activation energies for folding and unfolding in A8-35 versus detergent indicates that stabilization of A8-35-trapped OmpA against denaturation by urea is a kinetic, not a thermodynamic phenomenon.

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). ...

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. Major histocompatibility complex (MHC)2 class I molecules present antigenic peptides to cytotoxic T lymphocytes (CTL), which leads to the elimination of virus-infected cells. MHC class I molecules are heterotrimers consisting of a transmembrane heavy chain (H chain), a soluble subunit termed ␤ 2 -microglobulin (␤ 2 m), and a peptide ligand of 8 -10 residues. Assembly of class I molecules begins in the endoplasmic reticulum (ER), where the glycosylated H chain binds to the membrane-bound lectin chaperone calnexin (Cnx) and its associated thiol oxidoreductase, ERp57. At this early stage, the two highly conserved disulfide bonds within the H chain are formed, and the H chain assembles with ␤ 2 m. H chain-␤ 2 m heterodimers then enter a peptide loading complex (PLC), where class I molecules acquire peptides for display to CTL. The PLC consists of calreticulin (Crt), the soluble paralog of Cnx, an associated ERp57 molecule, a peptide transporter termed TAP, and tapasin, which is the nucleus of the PLC, bridging the interaction between class I heterodimers and the TAP peptide transporter. Once peptides are translocated into the ER by TAP, a subset bind to receptive H chain-␤ 2 m heterodimers with high affinity, triggering dissociation of class I molecules from the PLC and their subsequent export from the ER to the cell surface (1, 2).Although the functions of most of the participants in class I biogenesis are well understood, the details of how ERp57 functions in...