Amino acid sequence and tertiary structure of Cratylia mollis seed lectin

Gustavo A. de Souza2, Paulo S.L. Oliveira3,4, Stefano Trapani3, Ana Célia O. Santos5, José C. Rosa2, Helen J. Laure2, Vitor M. Faça2, Maria T.S. Correia6, Gisele A. Tavares3,4, Glaucius Oliva3, Luana C.B.B. Coelho6 and Lewis J. Greene1,2

2 Centro de Quimica de Proteínas and Depto de Biologia Celular, Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, SP, Brazil; 3 Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970, São Carlos, SP, Brazil; 4 Instituto de Química de São Carlos, Universidade de São Paulo, 13560-970, São Carlos, SP, Brazil; 5 Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife, PE, Brazil, and 6 Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Recif, PE, Brazil

Received on August 6, 2003; revised on August 25, 2003; accepted on August 26, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 References
 
Carbohydrate–protein interactions play a key role in many biological processes. Cramoll is a lectin purified from Cratylia mollis seeds that is taxonomically related to concanavalin A (Con A). Although Cramoll and Con A have the same monosaccharide specificity, they have different glycoprotein binding profiles. We report the primary structure of Cramoll, determined by Edman degradation and mass spectrometry and its 1.77 Å crystallographic structure and compare it with the three-dimensional structure of Con A in an attempt to understand how differential binding can be achieved by similar or nearly identical structures. We report here that Cramoll consists of 236 residues, with 82% identity with Con A, and that its topological architecture is essentially identical to Con A, because the C{alpha} positional differences are below 3.5 Å. Cramoll and Con A have identical binding sites for Me{alpha}Man, Mn2+, and Ca2+. However, we observed six substitutions in a groove adjacent to the extended binding site and two in the extended binding site that may explain the differences in binding of oligosaccharides and glycoproteins between Cramoll and Con A.

Key words: amino acid sequence / Cratylia mollis / crystallography / lectin / methyl-{alpha}-D-mannoside


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 References
 
The interaction of carbohydrates with proteins is a crucial step in many biological processes such as cell–cell recognition, adhesion, metastasis, bacterial and viral infection, and inflammation. Lectins belong to a group of homologous proteins that bind carbohydrates but are not enzymes or antibodies. They have been used as models for the study of the molecular basis of protein–carbohydrate interaction and specificity (Sharon and Lis, 1993Go, 1995Go). Leguminous lectins have a high level of primary structure identity and also present remarkable variations in carbohydrate binding. This specificity is demonstrable not only in terms of the recognition of monosaccharides but also by that of different oligosaccharides by lectins with the same nominal monosaccharide specificity (Sharon and Lis, 1990Go).

The structural basis for selective sugar recognition and binding has been identified by X-ray crystallography, which has revealed that primary binding sites of leguminous lectins are located in a shallow groove on the surface of a well-conserved structural fold (Derewenda et al., 1989Go; Loris et al., 1998Go). Selectivity is achieved primarily through the geometry of hydrogen bonds between sugar hydroxyl groups and protein main and side chains and through water-mediated hydrogen bonds (Weis and Drickamer, 1996Go). The affinity of legume lectins to complex carbohydrate ligands is enhanced by means of subsites in the lectin monomer that extend the monosaccharide binding groove (Rini, 1995Go; Loris et al., 1998Go).

Dioclea grandiflora lectin (DGL) and concanavalin A (Con A) are members of the Diocleinae subtribe and both bind mannose/glucose. Although they have 85% sequence identity (Richardson et al., 1984Go) and essentially the same topological architecture (Rozwarski et al., 1998Go), they have very different binding constants for complex carbohydrates. Isothermal titration microcalorimetry data indicate that the affinity constants of DGL and Con A for analogous trimannosides can differ from 3- to 4-fold, whereas the affinity constants for the biantennary pentasaccharide ß-GlcNAc-(1->2)-{alpha}-Man-(1->3)-[ß-GlcNAc-(1->2)-{alpha}-Man-(1->6)]-Man and longer chain analogs are 30-fold higher for Con A than for DGL (Gupta et al., 1996Go). These differences in the binding of specific oligosaccharides are also observed for other members of the Diocleinae subtribe (Dam et al., 1998Go) and result from differences in the extended binding site, which is continuous with the monosaccharide binding site (for a review, see Loris et al., 1998Go).

Cramoll is a lectin isolated from Cratylia mollis seeds (Correia and Coelho, 1995Go) from the same Leguminosae family and the same Diocleinae subtribe as Con A; like Con A, Cramoll is specific for mannose/glucose. Although they are closely related taxonomically, in immobilized form these two lectins bind different glycoproteins present in human plasma (Lima et al., 1997Go), and they also show differential binding to normal and transformed human mammary cells (Beltrão et al., 1998Go).

These apparent differences in the binding of glycoproteins have been the major stimulus for the present study, in which we determined both the primary and crystallographic structure of Cramoll. Our objective, then, was to compare Cramoll with Con A to obtain possible explanations for the differences mentioned. We report here that these lectins differ by 42/236 residues and that their topological architecture is essentially identical, as indicated by C{alpha} position differences. Furthermore, they have identical monosaccharide binding sites with identical hydrogen bond interactions between protein and methyl-{alpha}-D-mannoside (Me{alpha}Man). However, there are two residue substitutions in the extended binding site and six substitutions in a groove 15 Å away from the monosaccharide site, which could be responsible for the difference in the binding of glycoproteins.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 References
 
Sequence determination
Purified Cramoll was separated by reverse phase high-performance liquid chromatography (RP-HPLC) into three components, P1, P2, and P3, with Mr of 30,000 (intact protein, P3), 16,000 (P1), and 14,000 (P2), respectively. N-terminal sequence determination of the lighter components indicated that they corresponded to the N-terminal portion (P1) and the carboxyl terminal portion (P2) of the intact protein (P3). This has been described for other lectins of the Diocleinae subtribe and is the result of specific posttranslational peptide bond hydrolysis, which precedes the posttranslational synthesis of a new peptide bond (Carrington et al., 1985Go; Bowles et al., 1986Go). P1 and P2 were identified as residues 1–118 and 119–236 in intact Cramoll (see following discussion).

Because P1 and P2 were extremely insoluble, purified Cramoll containing P1, P2, and P3 was digested with trypsin or N-endoproteinase-Asp. Peptides were separated by RP-HPLC and subsequently submitted to automatic Edman degradation. The sequence data for 229 of 236 amino acids are given in Table I. The table also contains the elution time of the peptides from HPLC columns, N-terminal sequences of components P1 and P2 of Cramoll denoted ntP1 and ntP2, and a second internal sequence starting at residue 20 obtained during ntP1 sequencing, called ntP1*. The peptides yields (without correction for incomplete digestion, chromatographic losses, and sequencing losses) were 0.5% to 28.3%. The recovery of each PTH amino acid in picomoles during Edman sequencing is also given in Table I.


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Table I. Amino acid sequence data from N-terminal Cramoll light components (ntP1 and ntP2, 400 pmol) and Cramoll peptides released by trypsin and endoproteinase-N-Asp digestion

 
The peptides were ordered into three partial fragments on the basis of the overlap information available from N-endo-Asp peptides. When the tryptic and N-endo-Asp Cramoll peptides containing 229 residues were aligned with the primary structure of Con A, two regions that had not been sequenced were identified. These include the dipeptide LK, residues 115–116, and residues 168–172 (which are part of peptides T14 and D14) (Figures 1 and 2).



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Fig. 1. Electrospray ionization MS tryptic fingerprint of Cramoll. Peptides were identified on the basis of the amino acid sequence of the tryptic peptides determined by Edman degradation (inset). Peptide T14* ion (m/z 645.9 M + 2H) was predicted on the basis of sequence homology with Con A (m/z 659.7 M + 2H) and confirmed by CID. See Figure 3 for complete structure.

 


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Fig. 2. CID spectrum of parent ion 645.9 M + 2H (T14, Figure 1). The sequence obtained is VSNGSPQSDSVGR (residues 159–171), with an expected m/z of 645,3.

 


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Fig. 3. Amino acid sequence of Cramoll. Peptides are identified by letters that indicate the enzymes used for preparation: T indicates tryptic peptides and D endoproteinase-N-Asp peptides. Arrows indicate amino acid residues identified by Edman degradation. Arrows with a rounded head indicate that Edman automated analysis was interrupted before the end of the peptide. Bars indicate residues determined by MS. Asterisks indicate residues assigned on the basis of homology (residues 115 and 116).

 
Residues 115–116 were assigned on the basis of the alignment of Cramoll with the eight homologous lectin structures listed in Materials and methods. All belong to the Diocleinae subtribe, and all bind mannose/glucose. The eight sequences of these proteins were aligned from residues 110–120 (9/11 were identical), and the dipeptide LK at positions 115–116 was conserved in all eight lectins. The electron density maps of Cramoll, obtained from experimental diffraction data, were consistent with the presence of LK at positions 115–116.

Mass spectrometry (MS) was used to detect and sequence peptides T14 and D14. Figure 1 shows the peptide fingerprint of the tryptic digest of Cramoll. Nine of the ten peptide ions had m/z values that agreed ({Delta}<1.0 amu) with the tryptic peptides sequences, which were determined by Edman degradation (Figure 1, inset). By homology with Con A, we calculated the expected m/z for the Con A tryptic peptide in the same region as T14, VSSNGSPQGSSVGR, to be 659 for the double charged species. The ion with the closest m/z value detected in the Cramoll tryptic digest was 645 (M + 2H). The sequence of the Cramoll peptide T14 obtained from y series fragments was VSNGSPQSDSVGR (residues 159–172) (Figure 2). This peptide had not been recovered by HPLC from the tryptic digest. The parent ion (PI, 645 M + 2H) and two double-charged daughter ions, y8 (423.1) and y12 (596.3), also appear in the spectrum.

The expected sequence of peptide D14 was deduced by combining the amino acid sequence of T14 (residues 159–172, determined already) and T15 (residues 173–185, determined by Edman degradation, Table I) and carrying out a virtual digestion of T14/T15 with N-endo-Asp at the C-terminal side of aspartic acid. The expected m/z for the peptide DSVGRALYYAPVHIW was 874 for the double-charged species. When the m/z 874 ion of the N-endo-Asp fingerprint was submitted to collision-induced dissociation (CID), the partial sequence obtained was D(SV)G, which represents the y series, and (PV)HLxW, of the b series (data not shown). The determination of the composition and sequence of D14 was important to ensure that D14 was the overlap peptide of T14 and T15. Even with only partial characterization of the D14 sequence by tandem MS, this information and the m/z 874 M + 2H for this peptide, confirming but not demonstrating its expected composition, were sufficient to ensure that D14, DSVGRALYYAPVHIW, was an overlap sequence not previously identified.

The primary structure of Cramoll was deduced from sequence data summarized in Figure 3. Peptide T12 is the overlap peptide corresponding to residues 117–125 (SNSTADAQS). This demonstrates that the light components, P1 and P2, are fragments of the intact protein resulting from posttranslational proteolysis of residues 118–119 as has been described for Con A (Bowles et al., 1986Go). Peptide T20 is the result of partial trypsin hydrolysis, because three trypsin-sensitive sites (residues K184, K200, and R204) were not hydrolyzed by the enzyme. Thus peptide T20 provides the overlap information needed to align peptides T15, T16, T17, and T18.

The amino acid sequence proposed here for the lectin Cramoll consists of the direct assignment of 234/236 residues on the basis of Edman degradation and MS data. Residues L115 and K116 were the only residues assigned on the basis of homology with eight Diocleinae lectins.

Sequence alignment
Cramoll is closely related to the lectin from Cratylia floribunda (Craflor) (Cavada et al., 1999Go) in terms of its amino acid sequence, with 94% sequence identity. Both lectins have 236 residues in the single chain, whereas the other lectins of this tribe, such as Con A, have 237 residues. This is due to a deletion of a serine at position 161 of Con A, apparently with no effect on the overall structure. Cramoll has 82% amino acid sequence identity when compared to Con A (194/236 residues), and 42 substitutions.

The homology of Cramoll with other mannose/glucose lectins from the Diocleinae subtribe is demonstrated in Figure 4. Cramoll has 86% identity with Dioclea guianensis lectin (Diogu) and 85% identity with DGL. It is relevant to point out that the six amino acids (highlighted in Figure 4) that form the monosaccharide binding site are totally conserved in all five lectins.



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Fig. 4. Comparison of the primary structure of Cramoll with lectins from the Diocleinae subtribe. The primary structure of Cramoll was aligned with lectins from the Diocleainae subtribe: Craflor; two members of the genus Dioclea, Diogu and DGL; and Con A from jack bean. Shaded residues indicate the monosaccharide binding site and are conserved among these lectins. Asterisks indicate identity, and two dots indicate similarity.

 
Crystal structure of Cramoll
Quality of the model
The refined crystallographic model was obtained from a set of 25,553 (99.6% complete) unique reflections, extending up to 1.77 Å resolution. The mean redundancy of data was 3.8, with a discrepancy Rsym = 0.068, and the fraction of measured reflections with I/{sigma}(I) > 3.0 was 79.0%. In the highest-resolution shell (1.81–1.77 Å), diffraction data completeness was 98.1%, the average redundancy was 3.7, the average I/{sigma}(I) ratio was 4.9, and Rsym was 0.345. Space group was I222, with refined crystal cell parameters a = 63.4 Å, b = 78.0 Å, and c = 105.3 Å ({alpha} = ß = {gamma} = 90°).

The refined crystallographic model includes in the crystal asymmetric unit one monomer of Cramoll (236 amino acids), one calcium (II) ion, one manganese (II) ion, one molecule of Me{alpha}Man, and 179 structural water molecules. A total of 17 nonhydrogen atoms (pertaining to the side chains of Lys46, Gln135, Lys138, Arg158, Lys183, and Lys199) could not be localized in the experimental electron density maps and are missing from the polypeptide model. Alternate side chain conformations have been assigned to 22 amino acids. The calculated solvent content of the unit cell is 45.5%.

The crystallographic residuals of the final model are R = 14.2% and Rfree,5% = 17.0%. The average correlation coefficient between the theoretical amino acid electron density and the experimental (2D|Fo| - m|Fc|) Fourier map is 0.973 with a standard deviation of 0.027 and a minimum of 0.854. The lowest electron density correlation is found at the main chain position of residues 69–71, 117–121, 160–162, 183–185, and 203–204 and at the side chain position of Ser21, Asn82, Gln135, Asp151, Trp181, and His222, where the Fourier difference maps are not completely clear, likely due to some crystal disorder. Assignment of the amino acid sequence LK to residues 115–116 is consistent with the difference Fourier maps.

All residues in the crystallographic model have allowed {varphi}/{psi} values and 92.3% of the amino acids lie inside the core of the Ramachandran space. Only Ser117 has a G({varphi}/{psi}) factor (Laskowski et al., 1993Go) below -3.5. The average fine-packing quality-control value of the directional atomic contacts (Vriend and Sander, 1993Go) in the Cramoll structure is 0.108 with a z-score of 0.67 and only Gly223 shows a value of less than -2.5.

The tertiary structure
The monomer of Cramoll possesses the classical legume lectin fold (Loris et al., 1998Go) (Figure 5). It consists of three ß-sheets: a relatively flat six-stranded sheet, called the "back" ß-sheet; a seven-stranded curved "front" ß-sheet, which is packed against the back {alpha}-sheet; and a small five-stranded "top" ß-sheet that plays a major role in holding the two large sheets together. Four loops associated with the concave face of the front ß-sheet form a shallow depression, the monosaccharide binding site, where a molecule of Me{alpha}Man is found (Figure 6a). The two metal sites are found in the proximity of the monosaccharide binding site (Figure 6b).



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Fig. 5. Cramoll tertiary structure. Ribbon representation of Cramoll structure, demonstrating the location of Me{alpha}Man, Ca2+ ion, and Mn2+.

 



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Fig. 6. Stereo view of the Cramoll monosaccharide binding site (a) and metal binding sites (b). These sites are identical to the analogous sites in Con A.

 
Residues Asp10, Tyr12, Asn14, Asp19, Asp207, and Arg227 form the Cramoll calcium binding site, and the manganese binding site is formed by residues Glu8, Asp10, Asp19, His24, Ile32, and Ser34. The two metals interact with the polypeptide chain both directly and via coordination water molecules in an identical way already described for Con A (Hardman et al., 1982Go). One molecule of Me{alpha}Man in the monosaccharide binding site interacts through direct hydrogen bonds with Asn14, Leu99, Tyr100, Asp207, and Arg227 and through hydrophobic contacts with the side chains of Tyr12, Leu99, and Tyr100 in a way similar to that found in the Con A: Me{alpha}Man complex (Derewenda et al., 1989Go). These residues are fully conserved among the lectins of the Diocleinae subtribe (Figure 4).

The oligomeric assembly
There is a tetrameric arrangement of the Cramoll monomers, with 222 crystallographic point symmetry relating the four monomers in the tetramer. The tetrameric arrangement is equivalent to the tetrameric arrangement found in Con A and other Man/Glc-specific lectins (Prabu et al., 1999Go) and can be described as a dimer of dimers. A structural comparison with the Con A dimer results in 460 (97%) equivalent C{alpha}s having a positional difference less than 3.5 Å, with an overall rms deviation of 0.68 Å (1.08 Å for all C{alpha}s). Comparison of the tetrameric structures of Cramoll and Con A results in 920 (97%) equivalent C{alpha}s having a positional difference of less than 3.5 Å, with an overall rms deviation of 1.00 Å (1.13 Å for all C{alpha}s). The Cramoll dimer is formed by side-by-side interactions between the two back ß-sheets, resulting in a large 12-stranded ß-sheet, and by contacts between some residues from the loops interconnecting the front and the back ß-sheets. The calculated excluded solvent accessible area of the two monomers in the dimer is 2209 Å2, with a percentage of hydrophobic buried area of 51%. The two monosaccharide binding grooves are located at opposite sites in the dimeric arrangement. The Cramoll tetramer is formed by back-to-back packing of the large 12-stranded ß-sheets of the dimers. This results in an exclusion of 4391 Å2 of the solvent accessible surface of the two dimers and 61% of buried hydrophobic surface. Several water molecules are found at the dimer–dimer interface. Due to the slight concavity of the 12-stranded ß-sheets of the dimers, the tetramer presents a channel at its center, which is completely accessible to solvent.

The quaternary association of legume lectins is very variable (Prabu et al., 1999Go) and it has been proposed that this variability is associated with differences in the recognition of complex glycoconjugates and cross-linking processes. An extensive study was reported (Manoj and Suguna, 2001Go) in which correlations were found among primary structures, oligomeric association, and monosaccharide specificity of legume lectins. Five modes of dimeric arrangements have been described to date (Prabu et al., 1999Go). The Cramoll dimerization mode can be described as a "type II" dimerization mode, in contrast to various "type X" dimerization modes found in other lectins. The latter are characterized by the stacking of the back ß-sheets of the two monomers. The contacts between two monomers from opposite dimers in the Cramoll tetramer can be described as an X2-type monomer– monomer interface. One amino acid in the primary structure of legume lectins, corresponding to Ala176 in Cramoll, has been considered critical for either the type II or type X dimer formation (Manoj and Suguna, 2001Go). In lectins that form dimers of type II, this residue is found at the monomer–monomer interfac, and is invariably small (either Ala, Ser, or Thr). On the other hand, in lectins that form dimers of type X, this residue has a charged long side chain (Glu, Lys) that would hinder the formation of a type II dimer.


    Discussion
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 Abstract
 Introduction
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 Material and methods
 References
 
The identical binding of monosaccharides by the taxonomically related Cramoll and Con A and the differential binding of oligosaccharides in glycoproteins (Lima et al., 1997Go; Beltrão et al., 1998Go) suggest subtle structural differences between these homologous lectins. These considerations stimulated us to determine the amino acid sequence and crystallographic structure of Cramoll. Our initial objective was to compare Cramoll with Con A in an attempt to explain how oligosaccharide binding differences are achieved. We now report the primary structure of Cramoll and its crystal structure at 1.77 Å resolution.

Initially the structures of the two lectins showed 42 amino acid substitutions in 236 residues. When the crystallographic structure of Cramoll complexed with Me{alpha}Man was obtained, we observed that most of these substitutions were far from the monosaccharide and metal binding sites. In addition, Cramoll and Con A have nearly identical tridimensional structures, because positional least-squares comparisons of the Cramoll monomer structure with the Con A monomer (PDB entry 5CNA; 82% sequence identity) resulted in 231/236 equivalent residues having a C{alpha} positional difference below 3.5 Å and an overall rms deviation of 0.664 Å (0.995 Å for all C{alpha}s).

A single conservative amino acid substitution (Ile32Val), which does not modify the protein–metal interactions, is observed between the Cramoll and the Con A Mn2+ and Ca2+ binding sites. The metal binding sites are well conserved among the legume lectin structures and are indispensable for sugar binding. Metal binding is important for the correct folding of the loops that form the monosaccharide binding site by favoring the trans -> cis isomerization of Ala206–Asp207 in Cramoll and Ala207–Asp208 in Con A (Bouckaert et al., 1996Go). Amino acid residues involved in the interaction with the monosaccharide are fully conserved (Figure 4). Furthermore, the hydrogen bond distances in the monosaccharide and metal binding sites are practically identical, indicating that differences in binding observed between Cramoll and Con A do not result from significant differences in the architecture of the monosaccharide and metal binding sites. Recent data suggest that oligomeric assembly can be a relevant factor in differences in specificity between leguminous lectins (Prabu et al., 1999Go; Srinivas et al., 2001Go). However, the oligomeric arrangement of Cramoll in the crystal is tetrameric, analogous to the tetrameric structure of Con A.

To determine if substitutions in the neighborhoods of the monosaccharide binding site (the so-called extended binding site) (Naismith and Field, 1996Go) may be relevant to explain the reported binding differences between Cramoll and Con A (Lima et al., 1997Go; Beltrão et al., 1998Go), the Cramoll structure was superimposed on the crystallographic structures of Con A complexed with several oligosaccharides. Two regions containing differences were detected that could account for differences in oligosaccharide and glycoprotein binding between proteins.

Six substitutions are present in a groove at the opposite side of the extended binding site, approximately 15 Å from the Man/Glc binding site (Table II). These substitutions are located in a slightly deeper depression at the end of the long lateral crevice that, departing from the Man/Glc binding site, accommodates in Con A the biantennary pentasaccharide through the central reducing mannose and its O3-bound branch (Moothoo and Naismith, 1998Go). Figure 7 illustrates the surface representation of the groove in the two proteins. The two grooves are quite different both in shape and in the hydrophobic/hydrophilic properties of the surface. Up to now no lectin structure in complex with a ligand occupying this site is known, but it is reasonable to expect that this groove is important for interactions with more complex carbohydrates/glycoconjugates, and the structural differences mentioned are likely to give rise to rather different ligand specificities.


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Table II. Substitutions in the groove at the end of the extended binding site of Cramoll and Con A (see Figure 8)

 


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Fig. 7. Representation of the groove found at the end of the extended binding site of (a) Con A and (b) Cramoll. The Van der Waals lectin surfaces are illustrated. Surface color corresponds to carbon atoms (white), oxygen atoms (red), and nitrogen atoms (blue) of nonconserved residues. Conserved residues are represented in beige. The carbon atoms of the biantennary pentasaccharide bound to Con A is colored in light blue in (a). For the purpose of comparison, the biantennary pentasaccharide is reported at the equivalent position in the Cramoll representation (b). In (b), the Me{alpha}Man molecule bound to Cramoll is colored in green and is almost totally hidden in this view of the molecular structure.

 



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Fig. 8. Effect of the substitutions Ser168->Asp167 and Thr226->Gly225 on hydrogen bonding of Me{alpha}Man by Cramoll (a) and Con A (b). Illustrations of the extended binding site region where the substitutions Thr226->Gly225 and Ser168->Asp167 occur between Cramoll (a) and Con A (b). Color is white for the protein backbone cartoon representation and for carbon atoms, gray for oxygen atoms, and black for nitrogen atoms. Dashed lines in (a) and (b) represent H-bond interactions. In (a), the H-bond network of interactions between the O2 atom of Me{alpha}Man and the Cramoll structure is depicted; (b) is analogous to (a) for the Con A:Me{alpha}Man complex.

 
Another two additional substitutions are present in the extended binding site. Ser168 and Thr226 in Con A are replaced by residues Asp167 and Gly225 in Cramoll, respectively. For Cramoll, these substitutions result in the loss of a water-mediated hydrogen bond with the monosaccharide (Thr226 for Gly225) and, at the same time, in the gain of a new water-mediated interaction (Ser 168 for Asp 167) (Figure 8a and 8b).

The substitutions in the extended binding site are likely to be relevant to the interaction with oligosaccharides. Thermodynamic data indicates that DGL lectin possesses a 30-fold lower affinity for the biantennary pentasaccharide ß-GlcNAc-(1->2)-{alpha}-Man-(1->3)-[ß-GlcNAc-(1->2)-{alpha}-Man-(1->6)]-Man, when compared to Con A (Gupta et al., 1996Go). When the crystallographic structure of DGL was obtained complexed with a trimannoside (Rozwarski et al., 1998Go), it was suggested that substitutions of Thr226 and Ser168 in Con A for Gly226 and Asn168 in DGL explain the differences of affinity observed, because they result in loss of contact (Gly226) and steric hindrance (Asn168) with the NacGlc close to the monosaccharide binding site. Because Cramoll presents the same modifications (except for an aspartic acid at position 167 instead of asparagine in DGL), it is reasonable to conclude that Cramoll should have a lower affinity for biantennary pentasaccharide when compared with Con A.

The amino acid sequence, atomic coordinates and the experimental structure factors of Cramoll have been deposited with the Protein Data Bank (ID code 1MVQ) and will be released on publication of this article.


    Material and methods
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 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 References
 
Purification of Cramoll
Cramoll was isolated as described by Correia and Coelho (1995)Go. Briefly, an extract of C. mollis seeds was separated into two fractions with ammonium sulfate, 0–40% and 40–60% saturation. The 40–60% fraction was dialyzed against 0.15 M NaCl (F2) overnight at 4–6°C and affinity chromatographed on Sephadex G-75 (Sigma, St. Louis, MO) using a 1.9 x 70.0 cm column equilibrated with 0.15 M NaCl. Elution was performed with 0.3 M D-glucose, and the protein was dialyzed against 10 mM sodium citrate phosphate buffer, pH 5.5 (F3). F3 was chromatographed on a 1.5 x 31.0 cm column containing 50 ml CM-cellulose (Sigma) equilibrated with 10 mM sodium citrate phosphate buffer, pH 5.5, and eluted with a 0–0.4 M NaCl linear gradient.

Amino acid analysis
Amino acid analysis was carried out by the phenylthiocarbamyl derivative method (Bidlingmeyer et al., 1984Go) after acid hydrolysis with 6 N HCl containing 1% phenol in the vapor phase at 110°C for 22 h. A mixture containing 2.5 nmol of each amino acid (Standard H, Pierce, Rockford, IL) was used as standard and 4% 20 µl (100 pmol) was injected into a Picotag column (C18, 3.9 x 150 mm, Waters, Medford, MA), which was eluted with a gradient of solvent A (0.14 M sodium acetate containing 0.6% triethylamine, pH 5.7) and solvent B (acetonitrile:water 60:40), at 64°C (Rosa et al., 1999Go).

RP-HPLC peptide separation
Peptide separation was carried out using a Pharmacia HPLC system employing two pumps (model 2248) and a dual wavelength spectrophotometer (VWM2141) controlled by an LCC2252 gradient controller (Pharmacia-Amersham Biosciences, Uppsala, Sweden) and using a narrow bore Vydac C18 column (2.1 x 250 mm) (Separation Group, Hesperia, CA). Solvent A was 0.1% trifluoracetic acid (TFA) (v/v) and solvent B was acetonitrile: water:TFA (80:20:0.85). A linear gradient from 95% solvent A to 90% solvent B in 120 min at a flow rate of 0.2 ml/min was used.

Hydrolysis of Cramoll with trypsin and N-endoproteinase-Asp
Trypsin (modified sequencing grade from bovine pancreas; Boehringer Mannheim–Roche Applied Science, Mannheim, Germany) was incubated with the lectin at a 1/20 ratio (w/w) at 37°C for 72 h in 0.2 M ammonium bicarbonate buffer, pH 7.9. The reaction mixture contained 500 µg protein (20 nmol, determined by amino acid analysis) and 25 µg trypsin in a volume of 2.0 ml. The reaction was stopped with 20 µl 4% TFA. To prepare overlap peptides, 1 µg N-endoproteinase-Asp (Boehringer Mannheim) was incubated with 500 µg Cramoll (2.0 ml volume) in 50 mM sodium phosphate buffer, pH 8.0, at 37°C for 78 h. The reaction was stopped by freezing (Noreau and Drapeau, 1979Go).

After hydrolysis, 100 µl of the hydrolysate (1 nmol lectin) was submitted to RP-HPLC as already described.

Amino acid sequencing
The peptides produced by enzymatic hydrolysis of 1 nmol Cramoll were purified by RP-HPLC, and 100% of the recovered peptides were applied to a polyvinylidene difluoride membrane or glass fiber for automated sequencing using a Procise 491 Sequencer (Applied Biosystems, Foster City, CA). Yields were calculated on the basis of the average of the number of moles of PTH-amino acid recovered during the first three cycles. No corrections were made for digestion efficiency or HPLC losses.

MS
MS peptide fingerprints of the Cramoll digestion mixture were obtained with a triple-quadrupole mass spectrometer (Quattro II, Micromass, Waters, Manchester, England) equipped with an electrospray ion source. Aliquots containing 1 nmol of Cramoll equivalents of the trypsin and N-endo-Asp digestion mixtures were desalted using Vydac C18 resin eluted in 70% methanol containing 0.2% formic acid and infused continuously into the mass spectrometer at a flow rate of 20 µl/h. Sequence information was obtained by argon CID. Data collection and calculation of peptide masses were carried out using MassLynx software (Micromass).

Alignment of Diocleinae lectins
Alignment of the primary structure of Cramoll described here with other lectins from the Diocleinae subtribe: Craflor (Cavada et al., 1999Go), Diogu (Wah et al., 2001Go), DGL (Richardson et al., 1984Go), and Con A from Canavalia ensiformes (Cunningham et al., 1975Go; Wang et al., 1975Go) was carried out using CLUSTAL W software.

Assignment of residues 115–116
The expected dipeptide corresponding to residues 115 and 116 was not recovered from the tryptic digestion mixture. It was assigned the structure LK on the basis of homology with eight lectins of the Diocleinae subtribe using CLUSTAL W: Craflor (Cavada et al., 1999Go), Con A from C. ensiformis (Cunningham et al., 1975Go; Wang et al., 1975Go), Canavalia brasiliensis lectin (Sanz-Aparicio et al., 1997Go), Canavalia virosa lectin (Fujimura et al., 1993Go), Canavalia lineata lectin (Fujimura et al., 1993Go), DGL (Richardson et al., 1984Go), Diogu (Wah et al., 2001Go), and Dioclea lehmannii lectin (Perez et al., 1991Go), all mannose/glucose ligands.

Crystal structure determination
Purified Cramoll was cocrystallized with O1-Me{alpha}Man under conditions of microgravity for 9 days (NASA mission STS-84, May 1997) using a Vapor Diffusion Apparatus-2 and the precipitant conditions described by Tavares et al. (1996)Go. Elongated crystals in the orthorhombic system were obtained. They had well-defined faces and dimensions of 0.6 x 0.3 x 0.3 mm.

X-ray diffraction data were obtained from a single crystal at 279 K using the rotation monochromatic method. Data were collected in Campinas, Brazil, at the LNLS/CPr synchrotron beam line (Polikarpov et al., 1998Go) on a MAR345 image plate detector using an incident wavelength of 1.38 Å. A total of 61 diffraction images were recorded, each one corresponding to a crystal rotation of 1.5°. The diffraction images were processed using the HKL software package (Otwinowski and Minor, 1997Go). Data reduction was performed in space group I222.

The crystal structure was solved by the molecular replacement method using the deposited atomic coordinates of Con A (Naismith et al., 1994Go; PDB code 5CNA) and the AMoRe software (Navaza, 1994Go). The crystallographic model (1 monomer per asymmetric unit) was then refined iteratively in reciprocal and in real space using automated procedures and visual manipulation of the model. Reciprocal space refinement was initially performed using the X-PLOR software (Brünger, 1992Go) and then continued using the REFMAC5 program (Murshudov et al., 1997Go) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994), using a maximum-likelihood target with stereochemical restraints, a single TLS set of parameters for the whole model and individual restrained isotropic B-factors. A set representing 5% of the total experimental structure factors was excluded from the reciprocal space refinement target for cross-validation purposes. The program O (Jones et al., 1991Go) was used for the inspection of the (D|Fo| - m|Fc|) and (2D|Fo| - m|Fc|) difference Fourier maps and manipulation of the model. Water molecules were added automatically to the model on the basis of the difference Fourier maps and distance criteria using the program ARP/wARP version 5.0 (Lamzin and Wilson, 1997Go) from the CCP4 suite. The initial stages of the refinement were performed by mutating the molecular replacement model sequence based on the difference Fourier maps only. In the final stages, when the Cramoll sequence became available, the necessary corrections were introduced into the crystallographic model.

The stereochemical quality of the crystallographic model was constantly monitored during the refinement using the PROCHECK (Laskowski et al., 1993Go), WHATIF (Vriend and Sander, 1993Go), and O software. The model/experimental map correlation was calculated using the MAPMAN program (Kleywegt and Jones, Uppsala University, Sweden; available online at http://xray.bmc.uu.se/usf).

Accessible surface calculations were performed using the Lee and Richards (1971)Go algorithm implemented in the SURFACE program (CCP4 suite), using a 1.4 Å probe sphere. Structure superposition of Cramoll with other lectin structures available in the Protein Data Bank was performed using the program LSQMAN (Kleywegt,Uppsala University, Sweden). All molecular representations in the figures of this article were generated using PyMol (DeLano, DeLano Scientific, San Carlos, CA).


    Acknowledgements
 
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, the Howard Hughes Medical Institute, PADCT (FINEP), and Pronex (CNPq). We thank Richard C. Garratt for assistance and helpful discussion.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: ljgreene{at}fmrp.usp.br Back


    Abbreviations
 
CID, collision-induced dissociation; Con A, concanavalin A; Craflor, Cratylia floribunda lectin; Cramoll, Cratylia mollis lectin; DGL, Dioclea grandiflora lectin; Diogu, Dioclea guianensis lectin; Me{alpha}Man, methyl-{alpha}-D-mannoside; MS, mass spectrometry; RP-HPLC, reverse phase high-performance liquid chromatography; TFA, trifluoracetic acid


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 References
 
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