Journal of Biological Chemistry · 2002 · 85 citations · 41 references
Life SciencesBiochemistryMolecular BiologyProtein FoldingStructural BiochemistryStructural BiologyCalcium-binding ProteinsCarbohydrate RecognitionCargo ReceptorsEndomembrane SystemProtein StructureSecretory PathwayAnimal LectinsCarbohydrate Recognition DomainsCarbohydrate Recognition MotifsGlycoprotein TraffickingLectin InteractionsLectin RecognitionLectin-carbohydrate RecognitionLectins
p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 Å resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex β-sheet packed into a β-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi. p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 Å resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex β-sheet packed into a β-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi. endoplasmic reticulum carbohydrate recognition domain Protein Data Bank Secretory and membrane proteins undergo a quality control process that assures their proper folding, oligomerization, and maturation before exit from the endoplasmic reticulum (ER)1 (1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1064) Google Scholar). This is followed by the critical step of selection of cargo proteins to be exported from the ER to the Golgi complex via the ERGIC (ER-Golgi intermediate compartment). Export is then mediated by COPII vesicles (2Klumperman J. Curr. Opin. Cell Biol. 2000; 12: 445-449Crossref PubMed Scopus (110) Google Scholar). Although many proteins may become incorporated into COPII vesicles by default (bulk-flow transport), it is now generally believed that export is an active regulated process, whereby cargo molecules are first localized to ER exit sites and then selectively incorporated into COPII vesicles by one of several mechanisms (2Klumperman J. Curr. Opin. Cell Biol. 2000; 12: 445-449Crossref PubMed Scopus (110) Google Scholar, 3Balch W.E. McCaffery J.M. Plutner H. Farquhar M.G. Cell. 1994; 76: 841-852Abstract Full Text PDF PubMed Scopus (333) Google Scholar). Selective export of soluble proteins may occur by interaction with export receptors harboring motifs recognized by COPII coatomers. The p58 and ERGIC-53/MR60 proteins are the most commonly used markers for the ERGIC (4Saraste J. Palade G.E. Farquhar M.G. J. Cell Biol. 1987; 105: 2021-2029Crossref PubMed Scopus (125) Google Scholar, 5Schweizer A. Fransen J.A.M. Bächi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (375) Google Scholar). p58 (4Saraste J. Palade G.E. Farquhar M.G. J. Cell Biol. 1987; 105: 2021-2029Crossref PubMed Scopus (125) Google Scholar) and ERGIC-53 (5Schweizer A. Fransen J.A.M. Bächi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (375) Google Scholar) were originally identified as proteins reacting with antibodies prepared against Golgi membrane fractions, whereas MR60 was identified as a mannose-binding protein (6Pimpaneau V. Midoux P. Monsigny M. Roche A.C. Carbohydr. Res. 1991; 213: 95-108Crossref PubMed Scopus (24) Google Scholar). Subsequent cDNA cloning showed that ERGIC-53 (7Schindler R. Itin C. Zerial M. Lottspeich F. Hauri H.-P. Eur. J. Cell Biol. 1993; 61: 1-9PubMed Google Scholar) and MR60 (8Arar C. Le Carpentier V. Caer J.-P. Monsigny M. Legrand A. Roche A.-C. J. Biol. Chem. 1995; 270: 3551-3553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) were identical to each other and represented the human homologue of the rat p58 protein (9Lahtinen U. Hellman U. Wernstedt C. Saraste J. Pettersson R. J. Biol. Chem. 1996; 271: 4031-4037Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). p58/ERGIC-53/MR60 is a type I transmembrane, nonglycosylated protein with a lumenal domain, a transmembrane domain, and a short cytoplasmic domain. In cells, it is present as dimers and hexamers (9Lahtinen U. Hellman U. Wernstedt C. Saraste J. Pettersson R. J. Biol. Chem. 1996; 271: 4031-4037Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 10Lahtinen U. Svensson K. Pettersson R.F. Eur. J. Biochem. 1999; 260: 392-397Crossref PubMed Scopus (21) Google Scholar). The lumenal domain can be divided into two subdomains, an N-terminal carbohydrate recognition domain (CRD) (residues 31–285) and a membrane-proximal α-helical coiled domain (residues 290–460) (10Lahtinen U. Svensson K. Pettersson R.F. Eur. J. Biochem. 1999; 260: 392-397Crossref PubMed Scopus (21) Google Scholar). The cytoplasmic tail contains a KKFF sequence at its extreme C terminus that is essential for both ER exit and Golgi retrieval (11Kappeler F. Klopfenstein D.R.Ch. Foguet M. Paccaud J.-P. Hauri H.-P. J. Biol. Chem. 1997; 272: 31802-31808Abstract Full Text Full Text PDF Scopus (226) Google Scholar), meaning that p58/ERGIC-53/MR60 cycles between the ER and Golgi compartment (12Klumperman J. Schweizer A. Clausen H. Tang B.L. Hong W. Oorschot V. Hauri H.-P. J. Cell Sci. 1998; 111: 3411-3425PubMed Google Scholar). The identification of MR60 as a mannose-binding protein (8Arar C. Le Carpentier V. Caer J.-P. Monsigny M. Legrand A. Roche A.-C. J. Biol. Chem. 1995; 270: 3551-3553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and mutagenesis studies of ERGIC-53, in which the calcium-dependent binding of mannose to this protein was abolished (13Itin C. Roche A.-C. Monsigny M. Hauri H.-P. Mol. Biol. Cell. 1996; 7: 483-493Crossref PubMed Scopus (154) Google Scholar), support the suggestion that p58/ERGIC-53 serves as a mammalian intracellular lectin (14Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (133) Google Scholar). This proposition was also based on the similarity between ERGIC-53 and VIP-36, a membrane protein isolated from Madin-Darby canine kidney cells (15Fiedler K. Parton R.G. Kellner R. Etzold T. Simons K. EMBO J. 1994; 13: 1729-1740Crossref PubMed Scopus (193) Google Scholar), shown to bind mannose, and localized in the early secretory pathway (16Fullerkrug J. Scheiffele P. Simons K. J. Cell Sci. 1999; 112: 2813-2821PubMed Google Scholar, 17Hara-Kuge S. Ohkura T. Seko A. Yamashita K. Glycobiology. 1999; 9: 833-839Crossref PubMed Scopus (69) Google Scholar). It was proposed that p58/ERGIC-53 and VIP-36 constituted a new class of animal lectins (14Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Blocking the export of ERGIC-53 from the ER impaired but did not block export of the lysosomal enzyme cathepsin C and cathepsin Z-related protein (18Vollenweider F. Kappeler F. Itin C. Hauri H.-P. J. Cell Biol. 1998; 142: 377-389Crossref PubMed Scopus (134) Google Scholar). Furthermore, ERGIC-53 could be cross-linked to a cathepsin Z-related protein. The glycan structure binding to ERGIC-53 in the ER was suggested to be a nine-mannose form (Man9),i.e. the asparagine-linked core glycan from which three terminal glucose residues have been trimmed (19Appenzeller C. Andersson H. Kappeler F. Hauri H.-P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (273) Google Scholar). Efficient secretion of coagulation factors V and VIII requires a functional ERGIC-53 (20Moussalli M. Pipe S.W. Hauri H.-P. Nichols W.C. Ginsburg D. Kaufman R.J. J. Biol. Chem. 1999; 274: 32539-32542Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and mutations in the gene coding for ERGIC-53 cause a rare hereditary bleeding disorder, a combined deficiency of factors V and VIII (21Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Weatley M.A. Moussalli M. Hauri H.P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Taken together, these data strongly suggest that p58/ERGIC-53/MR60 functions as a lectin-like receptor involved in facilitating export from the ER of a subset of secretory glycoproteins (22Hauri H.-P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). As a first step toward a better understanding of how glycans on cargo molecules interact with the lectin receptor, we have determined the crystal structure of the CRD of p58. The structure reveals a link between leguminous and animal lectins and defines a new class of animal calcium-dependent lectins that function in the secretory pathway. The CRD of rat p58/ERGIC-53 (residues 31–285) was defined by a combination of sequence alignment, secondary structure prediction, and limited proteolysis. The CRD (including the N-terminal signal sequence comprising residues 1–30 and a 6xHis tag) was produced in insect cells using a baculovirus vector and purified as described elsewhere (23Velloso L.M. Svensson K. Lahtinen U. Schneider G. Pettersson R.F. Linqvist Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 536-538Crossref PubMed Scopus (8) Google Scholar). The region encompassing residues 286–478, which is not present in the construct whose structure is described here, is the oligomerization domain of p58/ERGIC-53 and is required for dimerization and formation of hexamers (10Lahtinen U. Svensson K. Pettersson R.F. Eur. J. Biochem. 1999; 260: 392-397Crossref PubMed Scopus (21) Google Scholar). Therefore, the CRD is monomeric in solution as assayed by native gel electrophoresis and gel filtration chromatography (data not shown). Crystals were grown by vapor diffusion from hanging drops containing equal volumes of a 10 mg/ml protein solution in 10 mm Tris-HCl, pH 7.5, 1 mmCaCl2, and well solution. The drops were equilibrated against 1 ml of well solution that consisted of 100 mmNa-HEPES, pH 7.25, 1.6 m Li2SO4, and 10 mm EDTA, as described previously (23Velloso L.M. Svensson K. Lahtinen U. Schneider G. Pettersson R.F. Linqvist Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 536-538Crossref PubMed Scopus (8) Google Scholar). The crystals belong to the orthorhombic space group I222, with cell dimensionsa = 49.6 Å, b = 86.1 Å, andc = 128.1 Å, and they have one monomer in the asymmetric unit (23Velloso L.M. Svensson K. Lahtinen U. Schneider G. Pettersson R.F. Linqvist Y. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 536-538Crossref PubMed Scopus (8) Google Scholar). X-ray data used for the structure determination were collected on a MAR 300mm Image plate detector mounted on a Rigaku R200 x-ray generator, operating at 50 kV and 90 mA at 110 K. Crystals were transferred to a solution containing 1.2m Li2SO4, 0.1 mNa-HEPES, pH 7.25, and 20% PEG400, allowed to equilibrate for a few seconds, and frozen in a stream of nitrogen. The high-resolution data set used for refinement was collected at beamline 711 at the MAX Laboratory (Lund, Sweden) synchrotron radiation source. The crystal was transferred to mother liquor containing 20% ethylene glycol before freezing. All data were processed with DENZO and SCALEPACK (24Otwinowski Z. Data Collection and Processing. SERC Daresbury Laboratory, Warrington, United Kingdom1993: 56-62Google Scholar). The structure was solved by multiple isomorphous replacement based on five heavy metal derivatives (Table I). Location of metal binding sites and phase calculations were performed using SOLVE (25Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) with a native data set collected at the home source as a reference. The initial electron density map calculated at 2.7 Å was of sufficient quality to allow automated model building of most residues in the structure and phase extension to 1.46 Å using wARP (26Perakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-464Crossref PubMed Scopus (2564) Google Scholar). This procedure was followed by multiple cycles of refinement in crystallography NMR software (CNS) (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; Scopus Google Scholar) using a and and using model building in S.W. M. Acta Crystallogr. Sect. A Crystallogr. 1991; PubMed Scopus Google Scholar). The cycles of refinement were performed using A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: PubMed Scopus Google Scholar) it to and and data have been at the for the and for the structure of the data and refinement Data and = = calculated for data between Å and the of each data set and for in to the of to 2.7 Å with from from of defined in in most in allowed in allowed = = = = = calculated for data between Å and the of each data set and for in to the As defined in in a new The crystal structure of the CRD of p58 was solved by multiple isomorphous replacement The initial electron density map was of sufficient quality to allow of most residues in the The model was to of and All data and refinement are in The model contains residues of p58, molecules and The region to residues 1–30 the signal which is secretion of the protein into the and is not present in the form of the protein. and the 6xHis between residues and are not in the electron density is also density for the residues of the construct and for residues of the p58 which are of a The CRD domain of p58 an and is of a and one of one and one against each a in a of the The terminus with two short and by a of This is by a and in other lectin we to it as in p58. This is the first of the It is followed by a and the first of the A and is between and the of the β-sheet here, the an into the opposite one before to the β-sheet and A followed by an of two is between and The then to the three The of is between the two with in the β-sheet and in the The and C of the are to each other in The two are to a concave surface of the on the side of the β-sheet and a convex surface on the side of the A cleft is formed by a between and of the by the and the and form a are in one between residues and at the of and the other between and the of the and on their to it been proposed (14Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (133) Google Scholar) that ERGIC-53 and VIP-36 a new class of animal lectins in the secretory pathway. It is that these proteins function as sorting receptors for glycoproteins the ER (22Hauri H.-P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). of this gene have been in several two other and have been identified as to p58/ERGIC-53 N. A. G. P. P. PubMed Scopus Google Scholar), D. T. A. and S. that their also act as cargo receptors for The fold is most conserved in proteins recognized they sequence and well in the region the CRD of the structure described against the data using the L. C. J. Mol. Biol. 1993; PubMed Scopus Google Scholar) and the G. J. Crystallogr. 2000; Scopus (193) Google Scholar) that the CRD of p58 is most similar to the leguminous lectins leguminous lectins have a core structure of a with a concave comprising and a convex comprising (Table the that the sequence between p58/ERGIC-53 and leguminous lectins are generally the core of these the and the secondary structure of p58 well with those of the leguminous lectins. The binding sites in these lectins are on the concave with most of the residues that in ligand binding from the between at the of the to were to that of p58 using the the default were of between the two of sequence is between the of Y. A. M. P. C. PubMed Scopus Google F. T. R. C. L. F. S. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google J.M. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google C. J. J. J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google M. Z. J. Mol. Biol. 1993; PubMed Scopus Google G. L. T. 9: Full Text Full Text PDF PubMed Scopus Google Y. S. M. M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google J. A. R. H. S. J.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google G. T. Y. T.C. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google The were to that of p58 using the the default were of between the two of The sequence is between the of in a new is also between p58 and other animal lectins as calnexin and as well as the domain of (Table is also a calcium-dependent lectin that resides in the ER and is involved in quality control mechanisms Y. S. M. M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). p58 and calnexin have a CRD the have similar and in the the sequence between the two proteins (Table which in the leguminous lectin are conserved and similar in both animal lectin to a family of animal lectins that are cytoplasmic J. A. R. H. S. J.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with p58, the and of are their similar to a family of proteins expressed in of in and to function as cell recognition Although for these molecules have not been it been proposed that they the binding fold and surface as the leguminous lectins to interact with cell surface molecules G. T. Y. T.C. J. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). p58 and in the region of the putative ligand binding and is similarity between residues to be involved in ligand binding in p58/ERGIC-53 and the residues of of p58/ERGIC-53 and from the animal reveals a of sequence The sequence is well the conserved residues and a that the core of the in between the the two and the in p58/ERGIC-53 and conserved in of this domain It is that the structure is conserved in other of conserved residues on opposite of the are that conserved residues are not to the core of the protein of p58. surface of p58. The two are by a in the of the and the concave and convex of the is and is by the of the putative ligand binding site. The are at the sequence the surface of p58. for the sequence are as described in the and for which mutagenesis studies have been are shown in and The are as described in A This was prepared using A. 1991; PubMed Scopus Google Image The form of p58/ERGIC-53 to mannose is not the ligand of (19Appenzeller C. Andersson H. Kappeler F. Hauri H.-P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (273) Google Scholar). it is that p58/ERGIC-53 on glycoproteins (6Pimpaneau V. Midoux P. Monsigny M. Roche A.C. Carbohydr. Res. 1991; 213: 95-108Crossref PubMed Scopus (24) Google Scholar, H.-P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). studies have and to be required for binding of ERGIC-53 to (13Itin C. Roche A.-C. Monsigny M. Hauri H.-P. Mol. Biol. Cell. 1996; 7: 483-493Crossref PubMed Scopus (154) Google Scholar). to the of in the are in the putative ligand binding site of the p58 structure with those of leguminous lectins. are between these binding site residues and in p58 are in similar to those of the and from and lectin in complex with mannose and we to these two proteins by their Y. A. M. P. C. PubMed Scopus Google Scholar, J.M. H. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). these residues the and also mannose in these The between residues and is in in p58, as in the leguminous lectins. This is essential for the of the binding site and for binding in these lectins Y. A. M. P. C. PubMed Scopus Google Scholar). between the binding sites from p58, and a of in p58 as with the in to a region between residues and of p58 of the side of Å from the in and and of residues and which are involved in in leguminous by and in p58, which it to bind the other residues of and that interact with mannose, and well on the residues and from p58, whereas the is of a and is Å from its in and data that ERGIC-53 as a cargo receptor for a subset of glycoproteins recognition of mannose residues in their (19Appenzeller C. Andersson H. Kappeler F. Hauri H.-P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (273) Google Scholar, H.-P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). this is a process, and glycoproteins have identical in the recognition of a subset of glycoproteins is most also in on to the recognized proteins other their surface calculations a on the surface of p58. A negatively charged is to the residues that are to be involved in and mannose binding The of a the in this but calculations suggest that it have a (data not shown). The sequence in the as well as of the negatively charged is that this region and its are for protein-protein the side of p58 opposite to the ligand binding is a surface patch of residues conserved in p58/ERGIC-53 It to the convex β-sheet in p58 and to its in p58/ERGIC-53 it may be involved in protein-protein interactions that are to as binding molecules in the formation of proteins two and of the p58 for complex the carbohydrate with the negatively charged on one side of for the cargo could bind to the conserved surface patch on the other side of This be of the interactions between calnexin and its using both its and lectin Y. S. M. M. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). p58/ERGIC-53 into dimers and hexamers in to other of this which are Therefore, of this surface patch in oligomerization also its in p58/ERGIC-53 In we have that the CRD of p58/ERGIC-53 is an of the of the lectin fold the quality control in the ER. The fold of the structure is a for The between the CRD domain of p58 and calnexin the of quality control mechanisms for the proper maturation of proteins in the ER. It is that this domain was present in before the of the and animal The structure here, with functional data on that the leguminous lectin fold is also used for recognition of carbohydrate in the proposition that these proteins a new class of animal calcium-dependent lectins. We to synchrotron radiation at the MAX Laboratory, beamline We for with data and refinement and for with
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