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• W1983708669 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 S. Acta Crystallogr. Sec. D. 1993; 49: 561-571Crossref PubMed Google Scholar), or even Ca2+ (11.Koenig S.H. Brewer C.F. Brown III, R.D. Biochemistry. 1978; 17: 4251-4260Crossref PubMed Scopus (48) Google Scholar, 12.Brewer C.F. Brown III, R.D. Koenig S.H. Biochemistry. 1983; 22: 3691-3702Crossref PubMed Scopus (41) Google Scholar). The S2 site is at 4.16 Å from S1 and binds Ca2+ but can also accommodate Cd2+ or Mn2+ (13.Marchetti P.S. Bhattacharyya L. Ellis P.D. Brewer C.F. J. Magn. Reson. 1988; 80: 417-426Google Scholar). Metal binding by ConA and the properties of the corresponding molecular species in solution have been studied in detail by Brewer et al. (for a review, see Ref. 14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar). Early nuclear magnetic resonance dispersion experiments indicated a high energy barrier (about 22 kcalm−1) separating the two conformers of ConA (15.Brown III., R.D. Brewer C.F. Koenig S.H. Biochemistry. 1977; 16: 3883-3896Crossref PubMed Scopus (160) Google Scholar). This high energy barrier and the consequently long equilibration times for the conformational change were attributed to the isomerization of the peptide bond between Ala-207 and Asp-208. Breweret al. (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar) conclude that ConA occurs in essentially two conformational states, which they called the locked, containing thecis peptide, and the unlocked state, containing a usualtrans peptide. Both states are in equilibrium with each other, but the equilibrium constant depends on the presence or absence of metal ions. In the absence of any metal ions, the dominant species is the unlocked state. In the presence of specific metal ions, the conformational equilibrium shifts completely toward the locked state. The locked, metal-bound form of ConA corresponds to the native Mn2+- and Ca2+-bound ConA as it is observed in its carbohydrate-free form as well as in a number of carbohydrate complexes. We set out to characterize by x-ray crystallography the different species on the metal binding pathways that have been observed in solution (Scheme FS1, according to Breweret al. (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar)) and to follow metal ion binding and conformational conversions in the crystalline state (SchemeFS2). To do so, we made use of the observation that Mn2+ can functionally replace Ca2+ (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar, 15.Brown III., R.D. Brewer C.F. Koenig S.H. Biochemistry. 1977; 16: 3883-3896Crossref PubMed Scopus (160) Google Scholar). In the absence of Ca2+, Mn2+ binds to S2. Mn2+ in the S2 site has a much larger dissociation constant (K d = 25 μm at pH 6.4) than Ca2+(K d = 0.3 μm at pH 6.0) and is in rapid equilibrium with the solvent (11.Koenig S.H. Brewer C.F. Brown III, R.D. Biochemistry. 1978; 17: 4251-4260Crossref PubMed Scopus (48) Google Scholar). As such, it can be much more easily extracted from the protein. Equally important for our work was the observation that when Mn2+ is used as a replacement for Ca2+, the conversion from the unlocked to the locked form is slowed down significantly, occurring on a time scale of hours or even days (at 5 °C) instead of minutes (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar). This allowed us to collect x-ray data on intermediates that occur only transiently or as minority species in solution.Figure FS2Soaking and co-recrystallization experiments to obtain the crystals used in this study. The subscripts sol and cryst stand for protein in solution and protein in single crystals, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ConA was purified, demetallized, and crystallized as described previously (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). One crystal of the resulting metal-free ConA in the unlocked form (U ConA) was used to collect a 2.05-Å resolution data set on beam line BW7B of the DESY synchrotron, Hamburg, Germany. These data were merged with low resolution data previously collected on our home source (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). UMn ConA was produced by soaking a single crystal of U ConA for 1 h in the mother liquor to which 7 mm MnCl2 had been added. The crystal was subsequently mounted, and data were collected immediately after, using a rotating anode generator. To prepare LMnMn ConA, crystals of metal-free ConA were soaked for 3 h in 60 μl of the mother liquor at pH 5 with the addition of 5 mm of Mn2+, resulting in LMnMnpH5.0 ConA. The crystals were then mounted in borosilicate capillaries and stored at 4 °C for at least 2 weeks. Crystals of LMnMn ConA were also produced by co-crystallization of metal-free ConA and Mn2+, resulting in LMnMnpH7.0 ConA. These crystals were grown in sitting drops of 75 μl composed of 20 μl of 17.3 mg/ml metal-free ConA, 50 μl of reservoir solution (2 m(NH4)2SO4, 60 mmCH3COONa, and 3% polyethylene glycol 5000 monomethyl ether, at pH 7), and 5 μl of 8 mm MnCl2, equilibrated against the precipitant solution. Isomorphous crystals of LMnCa ConA were produced under identical conditions as for LMnMnpH7.0 ConA, except that CaCl2 was also added. We found that L ConA could be produced after soaking crystals of LMnMn ConA in EDTA-saturated crystallization mother liquor. The mother liquor contained 2.1 m(NH4)2SO4 and 120 mmsodium acetate at pH 7.0 and was batch-treated with Chelex-100. The soaking solutions was refreshed several times to ensure a complete dialysis of Mn2+ out of the crystal and into the surrounding liquor. Data were collected for different soaking times ranging from 1 h to several months, as well as for different pH values of the soaking solution, decreased in steps of 0.2 from 7.0 to 5.0 with the acetate buffer. No significant differences were observed among this set of structures. Our studies showed that space group I222 was maintained under all conditions, even at pH 5.0. Crystals of L ConA produced after an EDTA soak of 2 months were further soaked in the mother liquor containing 100 mm calcium. Data were again collected on this LCaCa ConA crystal. All crystals were mounted in borosilicate capillaries. All tools that were used to manipulate crystals were washed extensively in deionized water and treated in batch with Chelex-100 beads before use to prevent any contamination of the protein with trace metals. After data collection, the metal content of every crystal was checked on a scanning electron microscope with an energy-dispersive x-ray analyzer to ensure the presence of the required metal ions and the absence of undesired ones. An anomalous data analysis has been performed on LMnMnpH7.0ConA crystals. In the case of U ConA, synchrotron radiation was used (beam line BW7B of the DESY synchrotron), whereas all other data were collected using a Rigaku RU200 rotating anode CuKαradiation. All regular data were collected using a MAR image plate detector data, integrated using DENZO, and scaled using SCALEPACK (16.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), with the exception of the data of LMnMnpH5.0 ConA collected on a FAST detector and processed using MADNESS. The anomalous data of LMnMn ConA were scaled using SCALA (17.Evans P.R. Joint CCP4 and ESF-EACBM Newsletter. 1997; 33: 22-24Google Scholar) and subsequently scaled and merged using ROTAVATA and AGROVATA (18.SERC Daresbury LaboratoryActa Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The statistics of the data collections are shown in Table I.Table IX-ray data collection and refinement statistics of the six ConA structuresSpeciesU pH 5.0UMn pH 5.0LMnMn pH 5.0LMnMn pH 7.0L pH 7.0LCaCa pH 7.0Protein Data Bank code1DQ21DQ41DQ51DQ61DQ01DQ1DetectorMAR IPMAR IPFASTMAR IPMAR IPMAR IPData analysis softwareDENZODENZOMADNESSDENZODENZODENZOUnit cell a(Å)60.5060.8963.2363.2063.0162.96 b(Å)84.2385.0187.4487.4187.2987.31 c(Å)91.0291.6289.2789.3088.8188.94Space groupP21212P21212I222I222I222I222Resolution10.0–2.058.0–2.98.0–2.08.0–1.926–1.725–2.15Number of observed reflections68,95645,99548,70890,119311,946262,158Number of unique reflections26,10811,61519,43519,67127,21513,517Highest resolution shell2.10–2.053.03–2.902.10–2.002.00–1.901.76–1.702.25–2.15R-merge0.0920.0920.0680.0880.0610.104(0.290)(0.392)(0.275)(0.241)(0.294)(0.375)Completeness (%)93.5 (91.8)96.2 (96.4)89.0 (81.2)87.1 (77.7)99.7 (99.9)99.4 (99.9)〈I/ς(I)〉11.2 (4.7) 6.0 (2.8)12.94 (3.65)16.53 (7.69)22.4 (5.7)14.58 (5.20)Reflections with I > 3ς(I) (%)76.3 (54.8)83.2 (52.1)74.9 (48.7)86.2 (74.3)88.7 (68.3)77.90 (56.5)R-value0.2320.2000.1890.1860.1810.182(0.334)(0.430)(0.279)(0.233)(0.301)(0.276)R-free value0.2790.2460.210ND0.2040.227(0.360)(0.415)(0.286)(0.312)(0.323)Bulk solvent correctionYesYesNoNoYesYesr.m.s. bond lengths (Å)0.0080.0080.0150.0120.0130.013r.m.s. bond angles (degree)1.51.63.62.91.71.8r.m.s. dihedral angles (degree)25.927.128.327.527.127.0r.m.s. impropers (degree)0.81.52.01.71.51.9Ramachandran plot (non-Gly, non-Pro) Most favorable82.980.688.088.588.986.5 Additionally allowed16.119.212.011.511.113.5 Generously allowed0.80.30.00.00.00.0 Disallowed0.30.00.00.00.00.0Number of water molecules1443510612518486ND, no R-free value was determined. No ς cut-off was used in the refinement. The highest resolution shell values are displayed between brackets. r.m.s., root mean square. Open table in a new tab ND, no R-free value was determined. No ς cut-off was used in the refinement. The highest resolution shell values are displayed between brackets. r.m.s., root mean square. The structures were solved by molecular replacement when necessary. Rigid body refinement and subsequent positional and individual B-factor refinements were performed without ς cut-off on the data using X-plor (19.Brünger A.T. X-PLOR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar), except for U ConA, which was refined by CNS (20.Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. GrosseKunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Starting coordinates for refinement were the 2.5-Å structure of metal-free ConA (Protein Data Bank code 1apn (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 1.85-Å structure of LZnCa ConA at pH 7.1 (Protein Data Bank code 1enr (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 structures. Except for LMnMnpH5.0 ConA, which was refined earlier, the refinement was started with a high temperature (3000 K) slow-cool stage to remove model bias and to uncouple the crystallographic R-factor and the R-free factor. The refinement was evaluated by cross-validation usingR-free (21.Brünger A.T. Method Enzymol. 1996; 277: 366-396Crossref Scopus (276) Google Scholar), and a bulk solvent correction was applied and updated throughout the refinement. For the addition of the metal ions, no metal-ligand bond distance restraints were employed, and the metal ions were given no charge. Water molecules were added only if 1) the water had at least one hydrogen bonding partner, 2) the positive difference density level was higher than 3 ς, and 3) the electron density of the putative water re-appeared after refinement in the 2F o − F c map at a level of at least 1 ς. The final refinement statistics are shown in Table I. To confirm the nature of the bound metal ions and to ensure that the sample was not contaminated by trace amounts of, mainly, calcium during crystallization or crystal handling, the identity of the metal ions in the co-crystals of LMnMn ConA was evaluated by means of anomalous dispersion of the Mn2+ions. Identification of the metal ions was a prerequisite to prove the substitution of manganese for calcium, since no distinction can be made between Ca2+ (18 electrons) and Mn2+ (23 electrons) on the basis of the electron density. Manganese has its K-absorption edge at the x-ray wavelength of 1.896 Å. The anomalous dispersion experiment was performed, however, at the wavelength of 1.5418 Å of Cu Kα radiation, widely used in laboratory x-ray sources, in combination with a MAR area detector. Data of LMnMnpH7.0 ConA were collected using the inverse beam strategy, and Bijvoet mates were treated separately to calculate anomalous-difference Patterson and Fourier maps. An anomalous-difference Patterson map has been calculated with the subroutine HASSP of the program HEAVY (22.Terwilliger T.C. Acta Crystallogr. Sec. D. 1994; 50: 17-23Crossref PubMed Google Scholar) by means of single atom, two atoms, and cross-peak searches. Starting from crystals of metal-free or U ConA, we attempted to produce and characterize by x-ray crystallography the different metal-bound and metal-free species observed by Breweret al. (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar) in solution (Scheme FS1). The ConA species that could be produced by a successive number of experiments involving the addition or removal of metal ions from crystals of ConA are shown in Scheme FS2. The statistics for the data collection and refinement of each of these crystals are summarized in TableI. In the case of the UMn ConA, data to only 2.9 Å could be collected because of the transient nature of this species, which spontaneously converts to LMnMn ConA. Therefore, short data collection times were necessary, although UMn ConA crystals diffract only weakly. It was not possible to flash-freeze the UMn ConA crystals, as earlier freezing experiments had already indicated that this inevitably leads to contamination with Ca2+. Consequently all crystals were exposed to x-rays at room temperature. Crucial to the isolation of the transient intermediate UMn ConA was the use of Mn2+ as a substitute for Ca2+ in S2; first, because the addition of both Mn2+ and Ca2+ to U ConA crystals results in cracking of the crystals, and second, because the binding of Mn2+ instead of Ca2+ in S2 decreases the time constant of the locking process from about 1 min to 0.5 h in solution at 25 °C (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar). The UMn ConA species ultimately converts to LMnMn ConA. A third advantage of using only Mn2+ is that Mn2+ can be extracted from the LMnMn ConA crystals due to the weak binding of Mn2+ to S2 (Scheme FS1). This allowed the production of crystals of L ConA, which occurs only as a minor species in solution (14.Brewer C.F. Brown III, R.D. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (60) Google Scholar) (Scheme FS2). The crystal structure of metal-free ConA (also called apo-ConA or demetallized ConA) has recently been described by us at a resolution of 2.5 Å (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). To obtain a more detailed picture of this conformational state of ConA, we used synchrotron radiation to collect data to 2.05 Å, the maximum resolution observed on beam line BW7B of the DESY synchrotron. The structure corresponds to the predominant unlocked species or U ConA found in solution and confirms the results of our previous studies on metal-free ConA. As in the earlier 2.5-Å resolution structure, the disordered metal binding loop Pro-13–Tyr-22 is not visible in the electron density map. Soaking of crystals of U ConA with MnCl2 initially leads to the binding of a single Mn2+ ion in the S1 site if the soaking time is sufficiently short and data are collected immediately afterward. The structure of the UMn ConA complex is essentially identical to those in UZn ConA and UCo ConA that were obtained in the same way and that have been described in detail before (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). Changes compared with U ConA are minor and restricted to the metal binding sites. In contrast to UZn and UCo ConA, UMn ConA is not a stable entity. In the time course of 2 weeks at 4 °C, it spontaneously converts to LMnMn ConA. UMn ConA also contains an extra Mn2+ ion bound to Asp-80 and Asp-82 at a crystal lattice contact. This binding site has not been observed in any ConA structure before, but is not considered to be of functional importance. When crystals of U ConA (space group P21212;a ≈ 60 Å, b ≈ 85 Å,c ≈ 91 Å) are soaked with MnCl2 for a longer period of time, they go through a disordered phase showing blurred reflections. After 2 weeks, the diffraction quality of the crystals has improved dramatically, and their space group and unit cell have changed to those of the native crystal form of ConA (I222;a ≈ 63 Å, b ≈ 87 Å,c ≈ 89 Å). During this transition, the ConA tetramers in the unit cell have rotated 14° relative to each other, and ConA has transformed from the unlocked to the locked form, with Mn2+ ions bound in both sites S1 and S2. The structural changes corresponding to locking are complex and involve thetrans-to-cis isomerization of the crucial Ala-207–Asp-208 peptide bond and the structural reorganization of a large portion of the protein (7.Bouckaert J. Loris R. Poortmans F. Wyns L. Proteins Struct. Funct. Genet. 1995; 23: 510-524Crossref PubMed Scopus (65) Google Scholar). The resulting structure, LMnMnpH5.0 ConA, is virtually identical to that of LMnCa ConA. The acidity of the crystallization solution did not prevent locking and allowed the conversion to space group I222. The conformation of the metal binding loop Pro-13–Tyr-22 and of the monosaccharide specificity loop Thr-97–Glu-102 loop are thus largely pH-independent in the presence of an excess of specific metal ions. LMnMn ConA that was prepared in solution from U ConA could also be crystallized at pH 7. LMnMnpH7.0 ConA is isomorphous with LMnMnpH5.0 ConA obtained by soaking of U ConA crystals. Therefore we can conclude that the transformation from unlocked to locked in the crystal is identical to that in solution. When we compare LMnMn ConA with LMnCa ConA, only minor distortions were found in the direct neighborhood of the metal ions (TableII). Equally at pH 5 or pH 7, we found slightly larger metal-ligand distances and a significantly larger intermetal distance. These are in agreement with a poorer stabilization of the metal binding sites and the larger dissociation constant of Mn2+ from the S2 site (Scheme FS1 (11.Koenig S.H. Brewer C.F. Brown III, R.D. Biochemistry. 1978; 17: 4251-4260Crossref PubMed Scopus (48) Google Scholar)). In the structure at pH 5, the temperature factor of Mn2+ at S2 is slightly higher than at S1, whereas in all other fully metal-bound ConA structures close to pH 7, the opposite is true (Table II). This is probably due to the lower binding constant of Mn2+ in S2 at pH 5 compared with at pH 7.Table IIMetal ligand distances (Å) at S1 and S2 and comparison with LMnCa ConA (37.Weisgerber S. Helliwell J.R. J. Chem. Soc. Faraday Trans. 1993; 89: 2667-2675Crossref Google Scholar)UMn1 pH 5.0UMn2 pH 5.0LMnMn pH 5.0LMnMn pH 7.0LMnCa pH 6.5LCaCa pH 7.0S1 Metal ionMn2+ (44)Mn2+ (45)Mn2+ (28)Mn2+ (15)Mn2+ (15)Ca2+ (33) Glu-8 OE22.74 (53)2.95 (52)2.34 (16)2.21 (8)2.23 (14)2.15 (27) Asp-10 OD22.83 (40)2.90 (56)2.23 (23)2.22 (7)2.13 (15)2.50 (28) Asp-19 OD12.47 (31)2.07 (15)2.26 (13)2.46 (41) His-24 NE22.31 (49)2.17 (46)2.27 (14)2.24 (6)2.25 (9)2.24 (25) Water O2.20 (18)2.47 (16)2.26 (14)2.40 (34) Water O2.30 (26)2.40 (18)2.13 (9)2.48 (57) Average2.63 (47)2.67 (51)2.30 (21)2.27 (12)2.21 (12)2.37 (35)S2 Metal ionMn2+ (30)Mn2+ (13)Ca2+ (10)Ca2+ (38) Asp-19 OD22.24 (31)2.56 (14)2.30 (11)2.45 (36) Asp-10 OD22.54 (23)2.55 (7)2.45 (15)2.26 (28) Asp-10 OD12.26 (21)2.22 (8)2.28 (10)2.36 (33) Asn-14 OD12.64 (28)2.48 (15)2.55 (15)2.89 (37) Tyr-12 O2.23 (28)2.36 (11)2.31 (10)2.38 (32) Water O2.45 (35)2.11 (15)2.37 (10)2.53 (39) Water O2.52 (35)2.45 (15)2.32 (12)2.64 (26) Average2.41 (29)2.39 (12)2.37 (12)2.50 (33) S1–S2 distance4.344.324.164.25Temperature factors (Å2) of these ligands are given in parentheses. UMn 1 and UMn 2 are the two monomers in the asymmetric unit of UMn ConA. Open table in a new tab Temperature factors (Å2) of these ligands are given in parentheses. UMn 1 and UMn 2 are the two monomers in the asymmetric unit of UMn ConA. The Mn2+ ions of LMnMnpH7.0 ConA show up unambiguously as the two highest peaks in the anomalous Patterson map, and their positions coincide exactly with the metal ion positions in the crystal structure of ConA. This implies that identification of manganese using CuKα radiation was possible on the short wavelength side, even far away from the absorption edge of the anomalous scatterer, although a difference in intensity of the reflections of only 3.83 electrons (23.Einspahr H. Suguna K. Suddath F.L. Ellis G. Helliwell J.R. Papiz M.Z. Acta Crystallogr. Sec. B. 1985; 41: 336-341Crossref Scopus (38) Google Scholar) is calculated for the two Mn2+ ions in LMnMn ConA. Data collected in an identical manner for our LMnCa ConA crystals, with a the" @default.
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• W1983708669 cites W1539796472 @default.
• W1983708669 cites W1563420813 @default.
• W1983708669 cites W1563862675 @default.
• W1983708669 cites W1979916844 @default.
• W1983708669 cites W1990031333 @default.
• W1983708669 cites W1991369439 @default.
• W1983708669 cites W1995017064 @default.
• W1983708669 cites W2001641653 @default.
• W1983708669 cites W2002940241 @default.
• W1983708669 cites W2009088712 @default.
• W1983708669 cites W2010118197 @default.
• W1983708669 cites W2012076267 @default.
• W1983708669 cites W2026562921 @default.
• W1983708669 cites W2043790697 @default.
• W1983708669 cites W2055800251 @default.
• W1983708669 cites W2056006636 @default.
• W1983708669 cites W2057168496 @default.
• W1983708669 cites W2059665556 @default.
• W1983708669 cites W2069679922 @default.
• W1983708669 cites W2084101844 @default.
• W1983708669 cites W2084971027 @default.
• W1983708669 cites W2086609868 @default.
• W1983708669 cites W2086704971 @default.
• W1983708669 cites W2093476537 @default.
• W1983708669 cites W2109280121 @default.
• W1983708669 cites W2118079337 @default.
• W1983708669 cites W2136339729 @default.
• W1983708669 cites W2144302597 @default.
• W1983708669 cites W2158381104 @default.
• W1983708669 cites W2314552957 @default.
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