The Structural Features of Concanavalin A Governing Non-proline Peptide Isomerization

Abstract

The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207–Asp-208, positioned in a β-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn2+ ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the β-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional β-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207–Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive thetrans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically. The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207–Asp-208, positioned in a β-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn2+ ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the β-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional β-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207–Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive thetrans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically. concanavalin A Locked state unlocked state Lectins are a structurally very diverse class of proteins that bind carbohydrates with considerable specificity but moderate affinities (1.Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1656) Google Scholar). Their ability to bind carbohydrates often depends on the binding of metal ions that mediate with the carbohydrate directly (the Ca2+-dependent animal lectins (2.Drickamer K. Curr. Opin. Struct. Biol. 1995; 5: 612-616Crossref PubMed Scopus (84) Google Scholar)) or indirectly (lectins from the Leguminosae family). Leguminosae lectins form a large family of plant lectins that succeed in covering a broad range of fine specificity toward oligosaccharides through subtle variations in length and sequence of five different loops A-E (3.Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (480) Google Scholar, 4.Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (193) Google Scholar, 5.Young N.M. Oomen R.P. J. Mol. Biol. 1992; 228: 924-934Crossref PubMed Scopus (107) Google Scholar). All Leguminosae lectins share the need for transition metal ion and calcium binding to stabilize the active conformation of these loops. The five amino acids involved in metal binding are fully conserved. These are a histidine (His-24 in ConA1) and a glutamate (Glu-8 in ConA) for the transition metal ion binding site S1, an asparagine for the calcium binding site S2 (Asn-14 in ConA), and two aspartate residues (Asp-10 and Asp-19 in ConA) bridging S1 and S2 by their carboxylate groups. All these five amino acids originate from two β-strands and their connecting metal binding loop (see Fig.2). Carbohydrate binding by Leguminosae lectins also involves a number of conserved residues. The monosaccharide binding site contains an Asn, Asp, Gly/Arg triad, originating from three loops: the metal binding loop C, loop A that follows the conserved cis peptide, and loop B, respectively. The conserved asparagine (Asn-14 in ConA) of the triad binds carbohydrate via its amide nitrogen but also ligates calcium through its amide oxygen. The aspartate (Asp-208 in ConA) is involved in a conserved non-proline cis-peptide bond that is in turn stabilized by calcium binding in S2. The third member of the conserved triad (Arg-228 in ConA) interacts both with the carbohydrate and via a water molecule with the metal ion in S2. Furthermore, an aromatic residue in the metal binding loop is essential for carbohydrate binding. On the other hand, the carbohydrate binding residues in the carbohydrate specificity-determining loop, or monosaccharide specificity-determining loop D, and loop E (3.Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (480) Google Scholar, 4.Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (193) Google Scholar, 5.Young N.M. Oomen R.P. J. Mol. Biol. 1992; 228: 924-934Crossref PubMed Scopus (107) Google Scholar) are not conserved. Neither are they involved in metal binding. All Leguminosae lectins are, thus, in the same way dependent on metal ion binding. Therefore the mechanism by which metal ion binding establishes the minimal structural requirements for carbohydrate binding merits a better understanding. Most studies have focused on the metal binding properties of Con A, because demetallization of ConA is reversible, in contrast to most other Leguminosae lectins. Demetallization of other closely related leguminous lectins such as lentil and pea lectin is often irreversible and leads to precipitation of these proteins (6.Paulova M. Ticha M. Entlicher G. Kostir J.V. Kocourek J. Biochim. Biophys. Acta. 1971; 252: 388-395Crossref PubMed Scopus (24) Google Scholar). As a consequence, ConA is the only family member for which crystal structures of its metal-free and partially metallized forms have been analyzed (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar,8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The binding of metal ions to ConA occurs at two adjacent sites located at the top of the dome-shaped molecule (Fig.1). In its native state, ConA binds a transition metal ion in site S1 and a calcium ion in site S2. The transition metal binding site (S1) of ConA preferentially binds Mn2+ but can also accommodate Ni2+, Co2+ (9.Emmerich C. Helliwell J.R. Redshaw M. Naismith J.H. Harrop S.J. Raftery J. Kalb-Gilboa A.J. Yariv J. Dauter Z. Wilson K.S. Acta Crystallogr. Sec. D. 1994; 50: 749-756Crossref PubMed Scopus (44) Google Scholar), Zn2+ (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), Cd2+ (10.Naismith J.H. Habash J. Harrop S.J. Helliwell J.R. Hunter W.N. Wan T.C.M. Weisgerber Acta Crystallogr. Sec. D. PubMed Google Scholar), or PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The S2 site is at from S1 and binds but can also accommodate Cd2+ or Mn2+ L. J. Scholar). binding by ConA and the properties of the species in have been in by a J. Struct. PubMed Scopus Google Scholar). a the two of ConA PubMed Scopus Google Scholar). This and the for the conformational to the isomerization of the peptide bond between and J. Struct. PubMed Scopus Google Scholar) that ConA occurs in two conformational which they the peptide, and the unlocked state, a are in with but the depends on the or of metal ions. In the of metal the species is the unlocked In the of metal the conformational toward the locked The form of ConA to the native and ConA as is in its form as as in a number of carbohydrate to by the different species on the metal binding that have been in to J. Struct. PubMed Scopus Google Scholar)) and to metal ion binding and conformational in the state of the that Mn2+ can J. Struct. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the of Mn2+ binds to S2. Mn2+ in the S2 site a at at and is in with the PubMed Scopus Google Scholar). As can from the for the that Mn2+ is as a replacement for the from the unlocked to the locked form is on a of or of J. Struct. PubMed Scopus Google Scholar). This allowed us to on that only or as minority species in and to the crystals in The and for in and in ConA and as (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). crystal of the metal-free ConA in the unlocked form ConA) to a on of the These with on (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). ConA by a crystal of ConA for in the to which been The crystal and a crystals of metal-free ConA for in of the at with the of of in The crystals in and at for at Crystals of ConA also by of metal-free ConA and in These crystals in of of of metal-free of and at and of against the solution. crystals of ConA as for that also that ConA crystals of ConA in The and at and with The to a of Mn2+ of the crystal and into the for different from to as as for different of the in of from to with the of studies that at Crystals of ConA an of in the on ConA All crystals in All that to crystals in water and in with to of the with the metal of crystal on a with an to the of the metal ions and the of been on In the of of the other a All a and Z. 1997; PubMed Scopus Google Scholar), with the of the of ConA on a and The of ConA and 1997; Scholar) and and and Crystallogr. Sec. D. 1994; 50: PubMed Scopus Google Scholar). The of the are in and of the ConA of of with bond bond Most of water in the The are between in a in the The are between The structures by replacement and and without on the Scholar), for which by J. M. T. Acta Crystallogr. Sec. D. 1998; PubMed Scopus Google Scholar). for the of metal-free ConA (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar)) for the unlocked structures and the of ConA at (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar)) for the locked for which the with a to and to the and the The by 1996; Scopus Google Scholar), and a and the the of the metal bond and the metal ions only the water at hydrogen bonding the and the of the water in the at a of at The are in the of the metal ions and to that the not by calcium or crystal the of the metal ions in the of ConA by of of the of the metal ions a to the of manganese for can between and Mn2+ on the of the its at the of The at the of of in in with a of ConA the and to and been with the of the Acta Crystallogr. Sec. D. 1994; 50: PubMed Google Scholar) by of two and from crystals of metal-free or to and by the different and metal-free species by J. Struct. PubMed Scopus Google Scholar) in The ConA species that by a number of the or of metal ions from crystals of ConA are in The for the and of of these crystals are in In the of the to only because of the of which to ConA crystals only not to the ConA as that leads to with crystals to at to the of the ConA the of Mn2+ as a for in because the of both Mn2+ and to ConA crystals in of the and because the binding of Mn2+ of in S2 the of the from to in at J. Struct. PubMed Scopus Google Scholar). The ConA species to A third of only Mn2+ is that Mn2+ can from the ConA crystals to the binding of Mn2+ to S2 This allowed the of crystals of which occurs only as a species in J. Struct. PubMed Scopus Google Scholar) The crystal of metal-free ConA or ConA) been by us at a of (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). a of conformational state of to to the on of the The to the unlocked species or ConA in and the of studies on metal-free As in the the metal binding loop is not in the of crystals of ConA with leads to the binding of a Mn2+ ion in the S1 site the is and are The of the ConA is to in ConA and ConA that in the same way and that have been in (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). with ConA are and to the metal binding In contrast to and ConA is not a In the of at to ConA also contains an Mn2+ ion to and at a crystal This binding site not been in ConA but is not to of crystals of ConA are with for a of they through a the of the crystals and their and have to of the native crystal form of ConA the ConA in the have to and ConA from the unlocked to the locked with Mn2+ ions in both sites S1 and S2. The structural changes to are and thetrans-to-cis isomerization of the Ala-207–Asp-208 peptide bond and the structural of a large of the (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). The is to that of The of the not and allowed the to The conformation of the metal binding loop and of the monosaccharide specificity loop loop are in the of an of metal ions. ConA that in from ConA also at ConA is with ConA by of ConA Therefore can that the from unlocked to locked in the crystal is to that in solution. ConA with only in the of the metal ions at or and a These are in with a of the metal binding sites and the of Mn2+ from the S2 site PubMed Scopus Google In the at the of Mn2+ at S2 is at S1, in other fully ConA structures to the is This is to the binding of Mn2+ in S2 at with at ligand at S1 and S2 and with ConA Helliwell J.R. J. Chem. Google Asp-10 Asp-19 Asp-19 Asp-10 Asp-10 of these are in and are the two in the of in a of these are in and are the two in the of The Mn2+ ions of ConA as the two in the and their with the metal ion in the crystal of This that of manganese on the from the of the a in of the of only H. K. G. Helliwell J.R. Acta Crystallogr. Sec. Scopus Google Scholar) is for the two Mn2+ ions in in an for ConA with a of to the Mn2+ not the of the Mn2+ ion in the A.J. Habash G. Hunter Raftery J. Helliwell J.R. A. H. in Scholar) in the for Mn2+ ion of ConA in the by at three different A.J. Habash G. Hunter Raftery J. Helliwell J.R. A. H. in at the of the at the and at a of the and ConA structures the Mn2+ ions as the In the of the Mn2+ to S1 with to S2 is In the of in S2 as the with a of Mn2+ of at H. K. G. Helliwell J.R. Acta Crystallogr. Sec. Scopus Google This the of Mn2+ in the S2 site of in with J. Struct. PubMed Scopus Google Scholar) and and studies J.H. J. PubMed Scopus Google Scholar). Crystals of ConA have been in their that with a of in the of the Mn2+ ions from both S1 and S2. The Mn2+ ion in S2 is that in against the at the different crystal structures that a of and ConA not of the a ConA that is to the for the Mn2+ ion in which by a water molecule as in ConA (see with for a locked is that metal ions in S1 and S2 The crystals of the metal-free ConA not convert to the of metal-free This not in a of with of the solution. a in of the not such a the the crystal as in the crystal and ConA from In crystal carbohydrate binding, which also the conformational toward the locked form J. Struct. PubMed Scopus Google Scholar). the crystal the locked state the carbohydrate binding site in its The ConA is very to ConA or between ConA and ConA are to the metal binding residues and are in S1 and are and from the metal ion in S1 and S2 in This with the in water is on the metal ion in the partially S1 The in of the water with the metal ion to form hydrogen or with the water that to metal The side chain of of the bridging carboxylate by This the S1 of the two water that ligates the metal ion in S1 and a hydrogen bond with the metal the of ConA in the metal binding sites water and in S1 and water and in S2. and are metal of S1, and and Asp-208 are of S2. All residues are and are for for and for In the locked both the S1 and S2 metal binding sites of metal-free ConA are and can metal ions. This us to investigate the of a locked species that contains only calcium and transition metal ion a species is to from studies PubMed Scopus Google Scholar), but S2 or both the of ConA crystals in a calcium leads to the of both metal binding with the side chain of Asp-19 to the calcium ions thus, the ligand water molecule to the S1 conformational changes are as the The of the calcium ions in S1 and S2 is as for The of structures us to the large between the unlocked and locked conformational from the metal ion The structural the locked and unlocked for locked unlocked structures and are from the state of the All residues by and to β-strands and their connecting loops. These β-strands are the loop, the Ala-207–Asp-208 peptide, and and and the monosaccharide specificity loop D. The with Thr-11 at its and the adjacent between and between the locked and unlocked conformation of ConA in S2 changes the conformation of the Asp-10 side chain and of the metal binding loop with the S2 and Asp-19 and the The bending of the β-strand the S2 site binding of a metal ion in S2 by the Asp-10 and leads to the of two conventional chain β-sheet hydrogen bond between and In unlocked the carboxylate of Asp-208 in between the β-strands and On the side of the monosaccharide specificity loop, Asp-208 two hydrogen with the amide side chain of the chain of Asp-208 a hydrogen bond with the Thr-11 These in unlocked ConA are in locked ConA by via side of Asp-208 with and located two β-strands The carboxylate in the of the Asp-208 carboxylate to Asp-208 in unlocked hydrogen in two the S2 interacts with the Thr-11 and toward the adjacent to the monosaccharide specificity loop, and interacts with the amide of by of the Mn2+ the of PubMed Scopus Google Scholar) that Mn2+ or can form a with ConA by the of both the S1 and S2 of the S2 in S1 is allowed with of the of the S1 in S2 is and a in the metal ion binding This is in the ConA both at and and by the The most for is that the of is in Chem. Scholar)) is with or Cd2+ (10.Naismith J.H. Habash J. Harrop S.J. Helliwell J.R. Hunter W.N. Wan T.C.M. Weisgerber Acta Crystallogr. Sec. D. PubMed Google Scholar) and to the for as a in the S2 ions Zn2+ and and bind in S2 but only in S1 (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). These the of Mn2+ from ConA crystals by studies with (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar) of such as the between two conformational species isomerization of the Ala-207–Asp-208 peptide bond and the of a locked ConA species in the of metal ions. In only of metal-free ConA is locked the is to ConA ConA is Mn2+ ions are from ConA in by J. Struct. PubMed Scopus Google Scholar), of the binding of Mn2+ to S2 with to ConA crystals are that are The the from to is but not by the crystal with the from to ConA is from to the crystal in the metal ion and carbohydrate binding in the and the of the crystal these crystal forces as for the in the in ConA have a different to The of ConA us to the two conformational locked and from the metal ion binding that the locked or turn to very in The same is for the of ConA structures in the unlocked The changes these two of structures are to in the metal binding In the unlocked and locked a of the of ConA crystals also allowed us to species from ConA This species from the and of Mn2+ from ConA crystals by in of Mn2+ and only that the of Mn2+ from S2 is of Mn2+ from S1, with the in and to a of ConA and These not a of the Asp-19 side in contrast to the ConA of metal ions. crystal structures the of but of the unlocked and locked forms in is the of the ConA species is to and the different species by J. Struct. PubMed Scopus Google Scholar) only is of This is the unlocked form metal ions in both S1 and S2. In the ConA the manganese in S1 is not at as also for and in the and (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google that Mn2+ binding in S2 of the unlocked form is an of in S1 is not that manganese binding in S2 in the unlocked state ConA) not by The crystal of ConA contains only a partially S1 site of residues and the S2 site is of Mn2+ to S1 in the unlocked state ConA) not to the of a S2 the Asp-10 and Asp-19 are to their with in and ConA (8.Bouckaert J. Poortmans F. Wyns L. Loris R. J. Biol. Chem. 1996; 271: 16144-16150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). of the metal ion in S1 the S2 site by for Asp-10 and Asp-19 to S2. This is but as an for the of the In the ConA Mn2+ binding in the S2 site which the toward the locked not to the directly in a because of the of the and the of the The the involves a or involves for the transition and by the of structural of the unlocked and locked of these are structures to steric This is of a mechanism in which the conformational changes with the isomerization of the Ala-207–Asp-208 peptide bond. The the transition contains S1 and S2 sites in with the peptide and the monosaccharide specificity loop of the unlocked state A metal ion is in S1 and in in with the of J. Struct. PubMed Scopus Google Scholar) on the ConA the of metal ions by by a also This the of the metal ions from ConA in to ConA that to This contains the metal binding sites from the peptide bond in the cis and the monosaccharide specificity loop of a metal ion in S2 is the to by and Asp-19 and the metal binding site of the locked form The β-strand S2 to the metal the conventional chain to chain β-sheet hydrogen bonding between and the adjacent a and the side chain of Thr-11 against the Ala-207–Asp-208 peptide bond Thr-11 a steric with Asp-208 between Thr-11 and Asp-208 between Thr-11 and Asp-208 A hydrogen bond form between Thr-11 and Asp-208 that the of steric between Thr-11 and the peptide thetrans-to-cis isomerization. to the isomerization, the Asp-208 side chain the and toward the monosaccharide binding the side chain of in to for of the Asp-208 carboxylate isomerization The of the β-strand and on the side of the cis peptide from the S2 and the conformation of the monosaccharide specificity loop by the isomerization from the locked to the unlocked state, metal ions are to the ConA the β-strand from the S2 site and This leads to the of the hydrogen bond between the Thr-11 and the carboxylate is In the side chain of Thr-11 the of the β-strand at The of Asp-208 into with and of Most the between residues and is by the of the β-strand The cis peptide is and the to and with a Ala-207–Asp-208 the peptide bond isomerization or of ConA can also by carbohydrate binding of by calcium binding. 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The of the lectins to the of the The of the isomerization the lectins from the of the in which they can to the against and 1995; PubMed Scopus Google Scholar) in the and in for the for of locked and unlocked metal-free the from the of of R. and as as the of and for their the on in D. from the of at the and from the are for their in the