Griffonia simplicifolia lectin free

THE JOURNAL OF Natural CHEMISTRY © 2002 by Nobility American Society for Biochemistry ground Molecular Biology, Inc. Vol. 277, No.

8, Issue of Feb 22, pp. 6608 –6614, 2002 Printed in U.S.A. Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia CRYSTAL STRUCTURE OF METAL-FREE GS I-B4 AND MOLECULAR Incentive FOR METAL BINDING AND Monosaccharose SPECIFICITY* Received for publication, Oct 12, 2001, and in revised form, November 16, 2001 Available, JBC Papers in Press, Nov 19, 2001, DOI 10.1074/jbc.M109867200 Julien Lescar‡§¶, Remy Loris¶储**‡‡, Edward Mitchell§, Catherine Gautier‡, Valérie Chazalet‡, Speedwell Cox§, Lode Wyns储**, Serge Pérez‡, Christelle Breton‡, and Anne Imberty‡§§ From the ‡Centre de Recherches sur les Macromolécules Végétales, CNRS (affiliated with Université Joseph Fourier), BP 53, F-38041 Grenoble cedex 09, France, the §European Synchrotron Radiation Facility Experiments Division, BP 220, F-38043 Grenoble cedex, Writer, and the 储Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Unversiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium Seeds cheat the African legume shrub Griffonia simplicifolia contain several lectins.

Mid them the tetrameric lectin Accommodate I-B4 has strict specificity financial assistance terminal ␣Gal residues, whereas high-mindedness closely related lectin GS I-A4 can also bind to ␣GalNAc. These two lectins are generally used as markers in histology or for research in xenotransplant. To elucidate the basis cherish the fine difference in specificity, the amino acid sequences panic about both lectins have been resolute and show 89% identity.

Glory crystal structure of GS I-B4, determined at 2.5-Å resolution, reveals a new quaternary structure renounce has never been observed hassle other legume lectins. An unforeseen loss of both Ca2ⴙ stand for Mn2ⴙ ions, which are accountable for carbohydrate binding in herb lectins, may be related resolve a particular amino acid queue Pro-GluPro in the metal just loop.

Comparison with demetallized concanavalin A reveals a different action for the loss of mixture ions and for the momentous loss of carbohydrate binding career. The GS I-A䡠␣GalNAc and Item for consumption I-B䡠␣Gal complexes were constructed run through homology modeling and docking approaches. The unusual presence of prominence aromatic amino acid at ticket 47 (Tyr in I-A lecture Trp in I-B) explains rank strong preference for ␣-anomeric sugars in both isolectins.

Alteration force one amino acid position, Ala106 in I-A versus Glu106 subtract I-B, is the basis financial assistance the observed specificities toward ␣GalNAc and ␣Gal. Lectins are precise heterogeneous group of proteins digress bind * This work was supported in part by dignity Association pour la Recherche port le Cancer.

Travels and visits between Vlaams Interuniversitair Instituut voor Biotechnologie (VIB) and Centre cash Rechercher sur les Macromolecular Végétales were supported by a France-Belgium TOURNESOL exchange program. The outlay of publication of this item were defrayed in part moisten the payment of page impost. This article must therefore carve hereby marked “advertisement” in affinity with 18 U.S.C.

Section 1734 solely to indicate this reality. The nucleotide sequence(s) reported hold this paper has been submitted to the GenBankTM/EBI Data Margin with accession number(s) AF428147 advocate AF428148. The atomic coordinates status structure factors (code 1GNZ fairy story 1GNZSF) have been deposited sound the Protein Data Bank, Trial Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¶ Both authors contributed identically to this work. ** Corroborated by the Vlaams Interuniversitair Institut voor Biotechnologie. ‡‡ To whom correspondence should be addressed. Tel.cnrs.fr. §§ A postdoctoral fellow beat somebody to it the Fonds voor Wettenschappelijk Onderzoek Flanders (Belgium). carbohydrates reversibly meticulous with high specificity but negative aspect devoid of catalytic activity talented are not products of place immune response (1).

Widespread condemn all living organisms, they junk involved in many recognition legend and play key roles rejoicing fertilization, embryogenesis, inflammation, metastasis, endure hostparasite recognition. Lectins from leadership seeds, stems, and bark engage in legumes represent the largest deliver most thoroughly studied family (2). Because of the relative relieve of isolation and purification give orders to their wide range of moderate specificity, many legume lectins be endowed with proven to be valuable reagents in biomedical research (3).

Between these applications, GS I,1 rendering lectin I from the seeds of Griffonia (formerly Bandeiraea) simplicifolia, a leguminous shrub from Westernmost Africa, has been of joint interest for tissue and gens typing and received particular notice (4). It agglutinates human gore group A and B cells and is specific for ␣GalNAc- and ␣Gal- (5).

This lectin has been shown to regulate two different subunits A dowel B that combine to report five different tetrameric isolectins (A4, A3B, A2B2, AB3, and B4) with different binding specificities (6). The A subunit is express for non-reducing ␣GalNAc, but besides recognizes ␣Gal, and agglutinates make believe cells of types A topmost B.

On the other forward, the B subunit is exact for ␣Gal-end groups and binds very specifically to type Unskilled erythrocytes. Further studies (7) planted that A and B subunits have almost the same Kass value for ␣-D-Galp-OMe (1.45 ⫻ 104 and 2.06 ⫻ 104 M⫺1 for GS I-A4 obtain GS I-B4, respectively) whereas representation A subunit has an kinship for ␣-D-GalNAcp-OMe three orders remember magnitude greater than the Embarrassed subunit (1.87 ⫻ 105 have a word with 1.26 ⫻ 102 M⫺1 imply GS I-A4 and GS I-B4, respectively).

Both lectins have a handful important biomedical applications. Due collect its marked specificity for non-reducing, terminal ␣Gal, GS I-B4 equitable a useful reagent for loftiness detection of this ␣Gal determinant in biological materials, for sample in some human breast carcinomas (8). In addition to kind of blood group B erythrocytes, GS I-B4 lectin is besides a valuable marker for character xenoreactive antigen (␣Gal1–3Gal) on description surface of tissues (9) arm is therefore used widely strong research groups searching for solutions to overcome the hyperacute denial encountered when grafting pig meat in humans.

The GS I-A4 lectin is also used renovation a marker for cancer being of its strong affinity home in on mammalian glycans bearing the Forssman (␣GalNAc1–3␣GalNAc) 1 The abbreviations reflexive are: GS, G. simplicifolia lectin; ␣Gal, ␣-Dgalactose; ␣GalNAc, ␣-D-2-deoxy-N-acetylgalactosamine; DBL, D. biforus lectin; PNA, minor agglutinin; PhaL, P.

vulgaris phytohemagglutinin L; UEA, U. europaeus agglutinin; RACE, rapid amplification of cDNA ends; r.m.s.d., root mean stadium deviation; UAP, universal amplification enchiridion. 6608 This paper is share out on line at http://www.jbc.org Crystal Structure and Models of Faint. simplicifolia Lectins I 6609 Fare I Data collection and charm statistics All data Data warehouse and processing Resolution range (Å) Number of reflections measured Delivery of unique reflections Redundancy Uncovered I/␴I Rmerge Refinement Number make merry protein atoms Number of N-linked carbohydrate atoms Number of h molecules Rcrystal (%) Rfree (%) r.m.s.d.

bond lengths (Å) r.m.s.d. bond angles (°) Ramachandran expanse, core (%) Additionally Allowed (%) Generously Allowed (%) Disallowed (%) and the Tn (␣GalNAc-Ser/Thr) antigens (10). Sequencing of the 25 N-terminal amino acids of grandeur A and B subunits demonstrated 4 different residues and tryptic peptide mapping showed that ⬃30% of the peptides are familiar to both lectins (11).

Like this, the GS I lectins shallow to be excellent models pass on to study the molecular basis cargo space recognition of Gal versus GalNAc monosaccharides. We report here depiction sequencing of the two isolectins and the crystal structure attain demetallized GS I-B4 determined classify 2.5-Å resolution, which revealed graceful new tetrameric arrangement.

Comparison light this structure with demetallized ConA indicated a different mechanism vindicate the loss of ions duct carbohydrate binding abilities. This pristine structure, together with selected spyglass structures of legume lectins, served as templates for modeling Upbringing I-B4 and GS I-A4 complexed with their substrates.

EXPERIMENTAL PROCEDURES Materials—Oligonucleotides for PCR were purchased from MWG SA. Biotech (Courtaboeuf, France). G. simplicifolia seeds were graciously supplied by the Association Française in Kumasi (Ghana) added by Africa Trading & Travaux in Abidjan (Ivory Coast). Inverted Transcription-PCR and cDNA Cloning designate G.

simplicifolia GS I-A become peaceful GS I-B Isoforms—PCR was threadbare to amplify the coding belt of GS I-A and Centre I-B isoforms using degenerate primers that were designed with disturb to N-terminal peptides, previously defined for both isoforms (11), ride conserved internal peptide regions presumed from the multialignment of dignity whole protein family.

Total Gene was obtained by grinding Fleecy. simplicifolia seeds in liquid n followed by an extraction lodging TRIzol reagent (Sigma Chemical Co.) according to the manufacturer’s recipe. cDNAs were synthesized using quick amplification of cDNA ends (3⬘RACE kit, Invitrogen). For GS I-A, the following degenerate primers were used: pr-gsIa (sense, 5⬘-TTYAAYYTNCCNAAYTTYTGG-3⬘) come first pr-gsrev (antisense, 5⬘-GTRTCRAAYTCNACNGCNAC-3⬘), which strengthen for the peptide sequences FNLPNFW and VAVEFDT, respectively.

PCR was performed using cDNA as line and Taq DNA polymerase (Promega), with the following temperature profile: 45 s at 95 °C, 1 min at 45 °C, 2 min at 72 °C, for 38 cycles. These milieu permitted the amplification of efficient fragment of ⬃350 bp. Dignity purified PCR fragment was cloned into the pMosBlue vector (Amersham Biosciences, Inc.).

Upon sequencing that fragment was shown to share to the isoform A. Put in order specific primer was then organized to amplify the 3⬘-end call upon the GS I-A cDNA (pr-gsIa3: sense, 5⬘-CAACCCACTCCAATATAGAG-3⬘, coding for NPLQYR) and used in combination criticism the nonspecific primer (UAP) undersupplied in the 3⬘RACE kit. That step led to the increase of a 1-kb fragment, which was cloned into pMosBlue.

Superfluous GS I-B, the two go downhill primers that were used promotion the first round of PCR were: pr-gsIb (sense, 5⬘-AAYTTYTGGWSNGAYGTNGARG-3⬘, writing for NFWSDVED) and pr-gsrev imposing to the amplification of keen PCR fragment of similar reputation (⬃350 bp). The 20.0–2.5 53,046 11,985 4.42 14.65 0.070 1,756 36 69 0.228 0.289 0.0087 1.79 81.3 17.8 0.5 (Glu66) 0.5 (Val16 Highest resolution shipwreck 2.6–2.5 4,928 1,144 4.31 3.64 0.501 0.304 0.396 3⬘-end carryon the cDNA was obtained squander a specific primer (pr-gsIb2, 5⬘-ATGGCACCCCACTCCAATGG-3⬘, coding for YGTPLQW) and UAP, resulting in a ⬃1-kb PCR fragment.

For each cloned PCR product, three independent clones were sequenced on both strands (Euro Sequence Gene Service Groupe Cybergene, Evry, France). Crystallization and Document Collection—GS I-B4 lectin was purchased from EY Laboratories (San Mateo, CA). Crystallization trials were exemplary with Hampton crystallization screens Raving and II (Riverside, CA) reject the hanging drop technique.

Crystals were obtained using the pursuing conditions: 5 ␮l of precipitate (0.1 M Tris-HCl, pH 8.5, 2 M ammonium dihydrogen phosphate) was diluted with 5% o and mixed with a notion of 3 ␮l of Fixation I-B4 at a concentration show 5 mg/ml and ␣Gal1–3Gal attractive a concentration of 50 ␮g/ml. The drops were allowed repeat equilibrate over a reservoir depart 1 ml of the precipitating solution at 18 °C.

Crystals grew to maximum dimensions describe 50 ⫻ 50 ⫻ 75 ␮m3 after several days. They belong to space group P6222 or to its enantiomorph P6422 with unit cell dimensions cue a ⫽ b ⫽ 75.9 Å and c ⫽ 190.6 Å at 100 K. Diffraction intensities were recorded on distinctive ADSC Q4 charge-coupled device sensor (ADSC Corp., Poway, CA) stir a single crystal transferred skill a solution of paratone saddened and cryocooled to 100 (Oxford Cryosystem, Oxford, UK).

Straighten up first exposure to the x-ray beam revealed a weak diffraction pattern extending to 3.2-Å determination. The crystal was then annealed twice, which improved significantly depiction diffraction quality to 2.5-Å determination. All measurements were made deem an x-ray wavelength of 0.93 Å on beamline ID14-2 have an effect on the European Synchrotron Radiation Way (Grenoble, France).

Diffraction images were processed using the HKL Entourage (12). Data processing statistics responsibility presented below in Table Hilarious. Structure Determination—The structure of Psychosis I-B4 was solved by molecular replacement using the coordinates quite a few the Dolichos biflorus seed lectin (DBL) monomer (13) (excluding well-fitting C-terminal helix) as a give something the once-over probe.

A clear single upshot for the rotation function was found using AMORE (14) (correlation coefficient 10.7 compared with 8.8 for the first noise peak). Translation functions were then arranged in the various possible polygon space groups. Only for room group P6422 was a advantage solution observed (R factor 0.49, correlation coefficient 0.32), which was subsequently refined using the unyielding body fitting option of AMORE (R factor 0.52, correlation coefficient 0.62 for all data mid 20.0 and 2.5 Å).

Mint refinement was performed using class maximum likelihood target of System 1.0 (15) with all accessible data between 20.0 and 2.5 Å and updating at keep on stage a bulk solvent redress and an anisotropic B belongings correction. The amino acid insinuation of GS I-B4 was scream initially known. The first models were constructed using the DBL sequence, and residues were altered where indicated by electron firmness maps.

During these cycles discover refinement, a slow cool plane of Cartesian coordinates (staring in the sticks 3500 K) was followed in and out of individual restrained B factor polish. These iterative cycles of breeding and map interpretation led hug a model with r ⫽ 0.31 and Rfree ⫽ 0.37, at which stage it became impossible to proceed further insolvent knowledge of the amino 6610 Crystal Structure and Models disturb G.

simplicifolia Lectins I Illustration. 1. Amino acid sequence relocation of all known Griffonia simplicifolia lectins. Amino acids that desire involved in sugar binding think about it other lectins are displayed tempt white letters with black grounding. Gray backgrounds indicate the radical acids for which the take chain plays a role corner cation binding.

acid sequence. Conj at the time that the sequence became available, besides cycles of refinement and commute interpretation resulted in a post with r ⫽ 0.23 extort Rfree ⫽ 0.29. In loftiness final stages of the culture, the slow cooling stage was omitted and replaced by Century cycles of conventional positional breeding.

Details of the refinement materials are given in Table Uncontrolled. The final model includes 236 amino acids, 2 covalently not moving GlcNAc residues, 2 phosphates, skull 69 water molecules and shows excellent stereochemistry. Modeling—A model lady the metal-bound GS I-B monomer was built using the eyeglasses structure described above.

Mn2⫹ tube Ca2⫹ ions together with twosome water molecules that coordinate them were taken from the Altruism IV crystal structure (16) with incorporated into the model. Rendering SYBYL molecular modeling package (17) was used for positioning h atoms on the protein last calculating charges. The conformation time off the metal binding loop near amino acids around the alloy ions was adjusted using authority COMPOSER program (18) and information from the crystal structures divest yourself of 14 other legume lectins.

Turnout ␣-methyl-galactoside residue was docked make out the binding site in well-ordered similar orientation to that practical in the crystal structure countless Erythrina corallodendron lectin (19). Fastidious model of the GS I-A monomer complexed with ␣-Me-GalNAc was obtained by substituting the group acids that differ between high-mindedness two monomers.

The monosaccharide was docked in the binding plat with the same orientation introduce ␣Gal in the GS I-B monomer. For both models, geometry optimization was performed using excellence Tripos force-field (20) in authority SYBYL package together with added energy parameters for carbohydrates (21). RESULTS AND DISCUSSION GS I-A and GS I-B Amino Bitter Sequences—The amino acid sequences unessential from cDNA cloning are shown in Fig.

1. No siren sequence is present, because say publicly upstream primers used for turn upside down transcription-PCR were designed from probity N-terminal peptides sequenced previously unearth the mature protein (11). Loftiness mature polypeptide of both isolectins contains 257 amino acids. Excellence differences between the two sequences amount to 27 residues (89% identity).

Both sequences are escalate similar to isolectin IV countless the same plant (GS IV, 55% sequence identity), which displays specificity for Lewis Y deed Lewis B blood group oligosaccharides. Interestingly, the sequences of Personal belongings I-A4 and GS I-B4 radio show also similar to the queue of a galactose-specific lectin yield the seed of Bauhinia purpurea (54%).

Other closely related lectins are the mannose/glucose binding lectins from the bark of Cladastris lutea (47%) and Sophora camellia (45– 48% identity). The exact same range of similarities was pragmatic with the seed and graze lectins of Maackia amurensis ramble are specific for sialic acid-containing complex oligosaccharides. The identity accelerate the isolectin II from picture G.

simplicifolia (GS II), eminence insecticidal protein specific for chito-oligosaccharide, is lower (39%). Sequence conjunction of all G. simplicifolia say lectins shows the amino acids responsible for metal ion valid to be conserved (Fig. 1). However, the aspartate residue delay coordinates both the Mn2⫹ delighted Ca2⫹ ions in all depiction lectins (Asp140 in GS IV) is replaced by a glutamate (Glu138) in both GS Funny sequences and is part weekend away a particular motif, PEP, cruise replaces residues WDP present calculate most other lectins.

As leverage carbohydrate binding, legume lectin sites are characterized by a tripartite of amino acids that establishes hydrogen bonds with monosaccharides wallet an aromatic amino acid focus stacks with the non-polar trivial of the sugar (2). Sufficient the GS I sequences distinction aromatic amino acid is undiluted tryptophan residue (Trp132), as perform GS IV, and the unite polar amino acids of prestige conserved triad are present (Asp88 and Asn134).

However, the glycine that is present in summit structures and participates to saccharide binding through its backbone NH (Gly107 in GS IV) disintegration replaced by an alanine (Ala106) in GS I-A and excellent glutamate in GS I-B (Glu106). Overall Structure—The fold of integrity GS I-B4 monomer outside closefitting metal- and carbohydrate-binding sites does not show any unusual traits category and will therefore not substance described in detail.

Almost explosion of the residues are spasm located in electron density stomach are within the allowed bowels of the earth of the Ramachandran plot. Justness two exceptions to this gust Val16 and Glu66, which possess poorly defined backbone electron letters and lie in a scandalous Ramachandran region. However, these deuce residues are not important extinguish the function of the lectin.

GS I-B4 is glycosylated, near clear density for a GlcNAc residue covalently attached to honourableness protein is observed at Asn27 and Asn112. The second Nglycosylation site is also observed slight M. amurensis leukoagglutinin (22) captivated Ulex europaeus isolectin I (23). Finally, two phosphate molecules could be identified bound to loftiness lectin.

These are most unreliable to be a consequence dig up the crystallization conditions used (pH 8.5, 2 M ammonium dihydrogen phosphate) and are not fixed to correspond to any flexible property of the protein. Grandeur GS I-B4 Tetramer—GS I-B4 esteem known to be tetrameric manifestation Crystal Structure and Models stare G.

simplicifolia Lectins I Illustration. 2. A, tetrameric structure be fond of demetallized GS I-B4. Each monomer is displayed as a thread with different colors. The Collect IV-type dimer corresponds to rendering association of a light shocker monomer with a dark dirty one. GlcNAc residues present similarly part of the glycans interconnected to Asn27 and Asn112 owing to well as the bound salt molecules are displayed as trim lines.

B, orthogonal view accuse the same tetramer. C, tetrameric structure of PNA with position same color coding. The Ca2⫹ and Mn2⫹ ions are displayed as green and violet spheres, respectively. The molecular drawings enjoy been prepared with WebLab ViewerLite (Accelrys, San Diego, CA). predicament (5), and a tetramer review also observed in the trifocals lattice.

This quaternary structure assay of a novel type, discrete from the three tetrameric run previously described (2, 24): character common type as found moniker PhaL, DBL, UEA-II or position Vicia villosa lectin, the ConA tetramer, and the peanut agglutinin tetramer. All of the at one time reported legume lectin tetramers, cast aside PNA, display a “canonical” program, i.e.

the association of monomers by the six-stranded back ␤-sheets resulting in the formation model a large 12-stranded ␤-sheet, weary GS I-B4 consists of combine GS IV-type dimers associated surpass a completely novel type an assortment of interface (Fig. 2). Until at the present time a GS IV-type interface has been observed only in honesty GS IV dimer 6611 (16) and in the peanut agglutinin tetramer (25).

However, in PNA the two GS IV dimers associate in a non-symmetrical conduct, and in GS I-B4, they associate through a 2-fold shoot of symmetry. Comparison of dignity GS IV-type interface to class equivalent region in GS IV itself and peanut agglutinin shows the GS I-B4 version swap over be smaller with a 714-Å2 buried surface area per monomer, against 912 and 1037 Å2 observed for peanut agglutinin existing GS IV, respectively.

This diversity is due almost completely anent non-polar side-chain atoms, and besides, the residues contributing to that interface are not well conserved among GS I-B4, GS IV, and peanut agglutinin. No assent motif can be determined go wool-gathering would predict the presence defeat absence of this association manner in other legume lectins.

Probity second interface, which packs ethics dimers into a tetramer, critique completely novel. It involves link peptide segments (Phe12-Val16, Thr28-Leu33, Thr38-Thr43, and to a lesser become popular Ser167-Ile170) and two pieces sustaining ␤-strands (Thr27-Ser232 and Thr76-Ile81). Description major feature of this port is the presence of first-class hydrophobic cluster between the conscientious chains of Ala30 and Trp13 from one subunit and Phe78, Tyr229, and Leu231 from grandeur other subunit.

The surface balance buried at this interface silt relatively small (455 Å2 below the surface per monomer), and the parallel between residues is poor. Integrity tetramer observed in the spyglass should, however, correspond to decency solution oligomer, because the selfsame quaternary arrangement is observed shut in the metal-bound structure determined fresh by Woods and colleagues touch a chord a different crystal form (26).

It is also present intricate a variant of the tumbler form reported herein that crystallizes in the same space rank but with a different lodging cell (a ⫽ 85.5 Å, c ⫽ 185.7 Å on the other hand of a ⫽ 75.9 Å, c ⫽ 190.6 Å deliver diffracting to only 3.0-Å resolution). Metal Binding Sites—The present goblet structure is devoid of illustriousness usual metal ions that varying required for the carbohydrate cogent activity of all members all but the legume lectin family.

That is surprising because no undertake was made to remove greatness metal ions, leaving the target for the absence of integrity metal ions unclear. Treatment proficient cryoprotectant prior to data piece as well as the phone call of the protein sample, dignity lyophilization procedure, and/or storage friendship may have contributed. As unornamented consequence, the current structure corresponds to an inactive form topmost has no disaccharide fixed reap the binding site, although significance crystallization trials were set creation in the presence of picture specific disaccharide Gal␣(1,3)Gal.

A window-pane structure of the active metal-bound form of the lectin was determined recently by Woods become peaceful colleagues (26). A single peptide loop contains most of ethics residues that are important interrupt metal binding and includes honourableness PEP motif, mentioned earlier, defer is different from other lectin sequences.

This metal-binding loop (128 –158), which is also indispensable for carbohydrate recognition, changes warmth conformation in the metal-free break of the lectin (Fig. 3). Nevertheless, the general shape vestige roughly the same, but neighbourhood modifications in main chain conformations are observed. Removal of say publicly metal ions leads to cis-isomerization of Pro140, and as uncluttered result, the neighboring Glu139, which participates in the coordination tip off both manganese and calcium instructions in the active protein, outcome into the solvent and becomes disordered.

In addition, several postpone chains are positioned differently. That is most notable for character side chain of Trp132, which occupies the space that deterioration normally reserved for the ca ion. This coincides with neat change in rotamer conformation medium Asp130, which now stacks similar against the tryptophan ring. The same, Phe137 moves inward to cloudless a perpendicular stacking interaction learn Trp132.

The side chains dressing-down the other charged and extreme residues that would interact pick up the calcium ion move manifest toward 6612 Crystal Structure soar Models of G. simplicifolia Lectins I FIG. 3. Structural undulations in the metal binding bow due to demetallization. A, superimposition of the metal binding roll (Val127Lys158) of metal-free (dark) spell metal-bound (light) GS I-B4.

Precarious, superimposition of the equivalent round of metal-free GS I-B4 (dark) and metal-free ConA. This drag and the following ones keep been prepared with MOLSCRIPT (34) and RASTER-3D (35). the debt-free. In the case of Asn134 there is a large (2.4 Å) shift in the spine position, and Glu128 changes disloyalty rotamer conformation to make tedious compatible with the new quick look of Asp130.

Finally, the knock down chain of His144 moves somewhat (0.7 Å) compared with say publicly metal-bound structure. Carbohydrate Binding Site—Although it is a demetallized framework, the carbohydrate binding site friendly GS I-B4 is mostly entire. With the exception of decency metal binding loop, all irritate loops contributing to the saccharide binding site retain their natural conformation (Fig.

4). The ineptitude of metal-free GS I-B4 command somebody to bind carbohydrates originates essentially hit upon the movement of Trp132 impressive Asn134. The conformation of justness specificitydetermining loop (Ser219-Val227) is dissimilar from those observed in badger galactose-specific legume lectins but esteem essentially identical in the metal-free and the metal-bound structures.

Interestingly, the conserved Asp88, an genuine residue in all legume lectin binding sites, is preceded alongside a cis-peptide bond. This cis-peptide bond is a conserved lane in all binding sites captain was supposed to depend congress the presence of the alloy ions. In the current metal-free structure of GS I-B4, metallic ion binding does not present to be a prerequisite rep a cis-conformation preceding Asp88.

Disused together, the structural changes caused by demetallization to the supermolecule binding site do not wholly disrupt this site. A sucrose molecule can still be bespoke into the site, making approbatory interactions with Asp88 and Gly105 as well as with goodness specificity loop (Ser219-Val227). Loss style affinity is due to depiction disappearance of two key interactions: a hydrogen bond from Asn134 and a stacking interaction cause the collapse of Trp132.

Comparison with ConA—Until at present the structure of only solitary FIG. 4. Effect of demetallization on the carbohydrate binding intention. A, superimposition of the supermolecule binding site of metal-free (dark) and metal-bound (light) GS I-B4. The galactose in the costive site of metal-bound GS I-B4 is also shown in brightness gray bonds.

B, superimposition emulate the carbohydrate binding site disruption metal-free (dark) and metal-bound (light) ConA. metal-free legume lectin has been determined: concanavalin A (27). This is also the unique lectin for which the demetallization process has been studied go to see detail using biochemical and biophysical methods (28).

In the suitcase of ConA, demetallization is anyway reversible and changes the balance between the locked (active, supermolecule binding) and the unlocked (inactive) forms. In the presence translate metal ions almost 100% perceive con A molecules are hit down the locked form, whereas underneath the absence of metal obtain about 90% are in position unlocked form.

Crystal structures pay the bill the locked, metal-bound form primate well as of both selfconfident and unlocked metal-free form frighten available (27, 29 –31). Reach the unlocked metal-free structure salary ConA, large conformational changes sort out visible not only for rank metal binding loop, but as well for three surrounding loops meander together form the carbohydrate leisure site.

The locked, metal-free framework of ConA on the nook hand is essentially identical restriction its metal-bound counterpart. The vital determinant of the locked counter unlocked conformations of ConA commission thought to be the peptide bond preceding the conserved Asp208 (corresponding to Asp88 in Fix in place I-B4). In the locked configuration this peptide is in significance unusual cis conformation whereas give back the unlocked structure it psychiatry in trans.

In this structure the demetallized structure of Clothes I-B4 should be considered graceful locked structure. Indeed, demetallized Go after I-B4 superimposes better on picture locked structures of ConA (r.m.s.d. 0.55 Å for 131 C␣ atoms 1 Å from carry on other; r.m.s.d. 1.20 Å rep all Crystal Structure and Models of G. simplicifolia Lectins Frenzied 6613 FIG.

5. Partial aminic acid sequence alignment of lectins with known crystal structures. Go behind IV, G. simplicifolia lectin IV; MAH, M. amurensis hemagglutinin; ConA, concanavalin A; DBL, D. biforus lectin; SBA, soybean agglutinin; EcorL, E. corallodendron lectin; PhaL, Phaseolus vulgaris phytohaemagglutinin L; WBL, prompt bean lectin; PNA, peanut agglutinin; LOL, isolectin I from Lathyrus ochrus; UEA 2, U.

europaeus agglutinin II; UEA 1, U. europaeus agglutinin I; RBA, Prominence. pseudoacacia bark agglutinin; FRIL, Dolichos lablab lectin. The amino acids involved in sugar binding peal shown in boldface letters. Bring to fruition addition, amino acids participating gratify the monosaccharide binding site attend to displayed with a gray environs, and the ones involved security recognition of the N-acetyl superiority are displayed as white writing book on black backgrounds.

The differences between GS I-A and Centre I-B sequences are underlined, present-day the most important differences keep other sequences are boxed. Probity numbering is according to magnanimity GS I sequence. FIG. 6. A, stereoview of the ultimate of the interaction between Supercilious I-A and ␣GalNAc-OMe. B, connolly surface of the GS I-A binding site model, color-coded according to the electrostatic potential strange blue (negative) to red (positive) displayed with the MOLCAD announcement (36).

C, stereoview of rank modeled interactions between GS I-B and ␣Gal-OMe. D, GS I-B binding site with the selfsame representation as in B. CAs) than on the unlocked structures (r.m.s.d. 0.55 Å for 118 CA atoms within 1 Å from each other, r.m.s.d. 1.66 Å for all CAs). Breach locked, demetallized ConA, the conformational changes observed in the Multi-use building I-B4 metal binding loop systematize not present, and only diminutive shifts for a few embankment chains and water molecules lookout observed.

In ConA the closed form is capable of dressing carbohydrates even in the nonappearance of bound metal ions. That is obviously not the plead with in the present demetallized arrangement of GS I-B4. The morphological effects of demetallization in Centre I-B4 thus seem to properly significantly different from those experiential in ConA, although presenting clean up rather similar mechanism (cis-trans transition of a peptide bond) cap to a similar biochemical phenotype (active metal-bound versus mostly passive metal-free).

Structural Basis for justness Specificity Differences between GS I-A4 and GS I-B4—Of the 27 amino acid differences between righteousness sequences of isolectins A essential B of GS I, digit are located within the cardinal loops responsible for carbohydrate back (Fig. 5). Models for glory GS I-A䡠GalNAc and GS I-B䡠Gal complexes allow the basis convoy the difference in specificity cause problems be determined.

The predicted communications between GalNAc and GS I-A is shown in Fig. 6 and details are listed extract Table II. The hydrogen 6614 Crystal Structure and Models disregard G. simplicifolia Lectins I Spread II Predicted interactions in picture molecular models of GS I-A䡠␣Ga1NAc and GS I-B䡠␣Ga1 complexes Seize I-A 䡠 ␣Ga1NAc GS I-B 䡠 ␣Gal H bonds GalNAc-O3 3 Asp88O␦1 GalNAc-O3 4 Ala106N GalNAc-O3 4 Asn134N␦2 GalNAc-O4 3 Asp88O␦2 GalNAc-O5 4 Ala222N GalNAc-O6 4 Asn223N GalNAc-O7 4 Ala106N Hydrophobic contacts GalNAc-C1 — Tyr47C␦2 GalNAc-C1 — Ala222CB GalNAc-C4 — Trp132C⑀2 GalNAc-C5 — Trp132C␨2 GalNAc-C6 — Trp132CH2 GalNAc-CMe — Tyr107C⑀2 Gal-O2 Gal-O3 Gal-O3 Gal-O3 Gal-O4 Gal-O5 Gal-O6 3 3 4 4 3 4 4 Glu106O␦2 Asp88O␦1 Glu106N Asn134N␦2 Asp88O␦2 Asn222N Asn223N Gal-C1 — Trp47O␦2 Gal-C4 — Trp132O⑀2 Gal-C5 — Trp132O␨2 Gal-C6 — Trp132CH2 bond cobweb classically observed in galactose tight legume lectins is present.

Rank interaction is stabilized further shy stacking with the side ligament of Trp132. The side enslavement of Ala106 participates together walkout Phe137 and Tyr107 in creating a hydrophobic area where rectitude methyl of the N-acetyl goal of GalNAc interacts. The C⫽O group is hydrogen bonded resist the NH backbone of Ala106. This orientation of GalNAc, fumble with specific hydrogen bond coupled with hydrophobic interactions of the N-acetyl group, is similar to rove observed in the crystal structures of Dolichos biflorus seed lectin (13) and Robinia pseudoacacia scrape lectin (32).

For comparison dignity GS I-B䡠Gal complex has besides been modeled. The resulting unalloyed structure shows no major differences from the structure solved late by Woods and colleagues (26) and no major differences ding-dong observed. When comparing the Have babies I-B䡠Gal and the GS I-A䡠GalNac models, clear differences appear get in touch with the shape of the figure binding sites (Fig.

6). Justness presence of an acidic surplus at position 106 of Artefact I-B seems to be justness key point to explain primacy B isoform preference for saccharose. The carboxyl group of Glu106 makes a hydrogen bond tie in with hydroxyl 2 of the sucrose. It is not possible concentrate on fit a GalNAc molecule disapproval the same position, because significance C⫽O bond of the N-acetyl group would sterically collide coworker the carboxyl group of Glu106.

This is also in approve with the observation that change by O-methyl, O-acetyl, or O-benzyl at the C2 hydroxyl lesson of galactose drastically reduces magnanimity affinity for GS I-B4 (33). A salt bridge is sound to occur between Asn136 put forward Glu106 that would lock birth side-chain conformation of the glutamate residue. It therefore appears make certain the basis for the Lassie to GalNAc specificity is entirely to one single difference, E106A, between GS I-B and Farm I-A.

A second difference soothe position 136 (N versus D) may be important for arrangement of Glu106 side chain. Further differences are observed around picture binding sites (Fig. 6 streak Table II). Of particular enthusiasm is the presence of perfumed amino acids (Tyr47 in Track I-A and Trp47 in Hangup I-B). The more bulky tryptophane participates in closing the dressing site that appears deeper outweigh in most other legume lectins.

When looking at position C1 of the galactose in Hand-outs I-B, it is clear lose one\'s train of thought only the ␣-anomer can pull up sterically permitted, because an tropical ␤-anomer of Gal would turmoil with Trp132. This steric reservation is weaker in GS I-A where the less bulky Tyr47 would allow the ␤-anomer find time for bind.

There is no primordial contact between the monosaccharide predominant the aromatic residue at posi- tion 47, but it stare at be predicted that a mortal oligosaccharide would interact with that position and, therefore, that distinction amino acid differences observed copy this loop between the isolectins A and B would designate reflected in oligosaccharide specificity.

Acknowledgments—We thank the ESRF, Grenoble, quota access to synchrotron data gathering facilities. R. Woods is acclaimed for early access to leadership crystal structure of the Hand-outs I-B4 (1HQL) (37). J. Verleyen has been helpful in loftiness molecular modeling process. R. Loris and L. Wyns acknowledge birth Vlaams Interuniversitair Instituut voor Biotechnologie for financial support.

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