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• W2017829148 abstract "It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The α- and β-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C−H···O-type hydrogen-bond(s) in protein−carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular. It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of 4-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The α- and β-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C−H···O-type hydrogen-bond(s) in protein−carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular. winged bean (basic) agglutinin isothermal titration calorimetry enthalpy-entropy compensation The recognition of sugar molecules by carbohydrate-specific proteins, lectins, involves the establishment of an organized set of interactions within the binding site. Hydrogen-bonding interactions are one of the most important factors of molecular recognition in lectin−sugar interactions, along with van der Waals forces, which, although rather weak (often contributing only a fraction of 1 kcal mol−1 for each pair of atoms), are frequently numerous and together make a significant contribution to binding (1Goldstein I.J. Hayes C.E. Adv. Carbohydr. Chem. Biochem. 1978; 35: 127-340Crossref PubMed Scopus (1343) Google Scholar,2Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar). Despite the differences in the lectin folds and their modes of sugar binding, the specificity for the recognition of galactose is determined by interactions involving the C-4 locus of the saccharide (1Goldstein I.J. Hayes C.E. Adv. Carbohydr. Chem. Biochem. 1978; 35: 127-340Crossref PubMed Scopus (1343) Google Scholar, 2Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar, 3Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (188) Google Scholar, 4Elgavish S. Shaanan B. Trends Biochem. Sci. 1997; 22: 462-467Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 5Vijayan M. Chandra N. Curr. Opin. Struct. Biol. 1999; 9: 707-714Crossref PubMed Scopus (229) Google Scholar). Stereochemical evidence is emerging for a distinct sugar binding specificity-dependent distribution of hydrogen bond donors vis à vis the acceptors in the combining site of lectins (4Elgavish S. Shaanan B. Trends Biochem. Sci. 1997; 22: 462-467Abstract Full Text PDF PubMed Scopus (218) Google Scholar). The C-4 locus of the monosaccharide within the primary binding site of galactose/N-acetylgalactosamine-specific lectins has hitherto been considered to be absolutely invariant.Though belonging to different families, jacalin, a Moraceaemember, and WBA1 I, aLeguminosae member, both are galactose/N-acetylgalactosamine-specific lectins (6Sastry M.V. Banarjee P. Patanjali S.R. Swamy M.J. Swarnalatha G.V. Surolia A. J. Biol. Chem. 1986; 261: 11726-11733Abstract Full Text PDF PubMed Google Scholar, 7Khan M.I. Sastry M.V. Surolia A. J. Biol. Chem. 1986; 261: 3013-3019Abstract Full Text PDF PubMed Google Scholar). Whereas jacalin displays a β-prism tertiary structural fold wherein a unique post-translationally generated N-terminal glycine residue serves as a critical determinant of galactose specificity (8Sankaranarayanan R. Sekar K. Banerjee R. Sharma V. Surolia A. Vijayan M. Nat. Struct. Biol. 1996; 3: 596-603Crossref PubMed Scopus (225) Google Scholar), WBA I contains a legume lectin fold (9Prabu, M. M., Sankaranarayanan, R., Puri, K. D., Sharma, V., Surolia, A. (1998) J. Mol. Biol. 76, Vijayan, M., and Suguna, K. 2, 787–796Google Scholar). During the course of mapping and establishing the hydrogen bond donor-acceptor relationship of the primary combining site of galactose-specific lectins (10Swaminathan C.P. Gupta D. Sharma V. Surolia A. Biochemistry. 1997; 36: 13428-13434Crossref PubMed Scopus (23) Google Scholar), unexpectedly, we have discovered that the 4-methoxy derivative ofd-galactopyranoside (4-methoxygalactose) binds, with affinities comparable with that of methyl-α-galactose, to theMoraceae lectin jacalin and the Leguminosaelectin WBA I but not to the related WBA II. 2Srinivas, V. R., Acharya, S., Rawat, S., Sharma, V., and Surolia, A. (2000) FEBS Lett. 474, 76–82.2Srinivas, V. R., Acharya, S., Rawat, S., Sharma, V., and Surolia, A. (2000) FEBS Lett. 474, 76–82.RESULTS AND DISCUSSIONThe ITC experiments directly detected the evolution of heats of binding when fixed aliquots of 4-methoxygalactose solution were added into either jacalin solution (Fig.1 A) or WBA I solution (Fig.1 C). These data, analyzed by iterated non-linear least squares fitting procedures, were found to best fit the simplest identical site model indicating that both the lectins bind to 4-methoxygalactose (Fig. 1, B and D). The reactions were driven by dominant favorable enthalpic contributions (Table I). These results, contrary to the usual expectation of the indispensability of the unsubstituted C-4 hydroxyl of the saccharide, clearly demonstrate the capability of 4-methoxygalactose to bind to both WBA I as well as jacalin. This reflects the existence of 4-methoxygalactose binding ability in galactose/N-acetylgalactosamine-specific lectin members from at least two unrelated families. To test whether 4-methoxygalactose displays a promiscuous binding to other lectins with similar monosaccharide specificity, the binding of 4-methoxygalactose to WBA II, a dimeric acidic lectin sequentially and structurally similar to WBA I, was tested. The results of such an ITC experiment indicate no binding of 4-methoxygalactose to WBA II (Fig. 1, E andF). That the non-binding of 4-methoxygalactose to WBA II was not due to loss of activity of WBA II was confirmed by testing the binding of 2′fucosyllactose to WBA II; the thermodynamic parameters obtained, K b = 1.0 × 105 and ΔH b 0 = 43.2 kJ mol−1 at 298.2 K, agreed very well with previously published values (12Srinivas V.R. Singha N.C. Schwarz F.P. Surolia A. Carbohydr. Lett. 1998; 3: 129-136Google Scholar). In contrast, none of these galactose/N-acetylgalactosamine-specific lectins, WBA I, WBA II, or jacalin, bound 4-methoxyglucose. The inherent inability of WBA II to recognize 4-methoxygalactose is perhaps due to dominant steric factors around its specificity-determining C-4 locus. The ability of 4-methoxygalactose to bind to WBA I as well as jacalin, together with its inability to bind to WBA II, is consistent with the formation of favorable interactions from the vicinity of the C-4 locus of 4-methoxygalactose with the corresponding binding site residues in WBA I and jacalin. It appears that the disposition of amino acid residues in the vicinity of the C-4 locus of 4-methoxygalactose within the binding sites of WBA I and jacalin permits the efficient binding of 4-methoxygalactose by WBA I and jacalin. That 3-methoxygalactose and 6-methoxygalactose do not bind to either WBA I (10Swaminathan C.P. Gupta D. Sharma V. Surolia A. Biochemistry. 1997; 36: 13428-13434Crossref PubMed Scopus (23) Google Scholar) or jacalin 3C. P. Swaminathan and A. Surolia, unpublished results. indicates that this unusual binding of 4-methoxygalactose by WBA I and jacalin is due to specific interactions. In addition, WBA I−4-methoxygalactose as well as jacalin−4-methoxygalactose complexes saturated with 4-methoxygalactose did not bind to a molar excess of exogenously added methyl-α-galactose, suggesting that 4-methoxygalactose was not bound to a site other than the primary combining site of either jacalin or WBA I.Table IThermodynamic quantities from ITC for binding of 4-methoxygalactose to galactose/N-acetylgalactosamine-specific lectinsLectinTK b−ΔG 0 b−ΔH 0 b−TΔS 0 b−ΔS 0 bK× 10 −3 M −1kJ mol −1J mol −1 K −1Jacalin278.238.94 (± 0.25)24.4537.24 (± 0.25)12.7946.0283.233.41 (± 0.33)24.5339.87 (± 0.23)15.3454.2288.229.41 (± 0.36)24.6541.25 (± 0.22)16.6057.6293.221.75 (± 0.17)24.3542.81 (± 0.28)18.4663.0298.216.12 (±0.21)24.0243.57 (±0.19)19.5565.6303.214.56 (± 0.34)24.1646.79 (± 0.32)22.6374.6WBA I278.215.18 (± 0.21)22.2721.07 (± 0.51)−1.20−4.3283.212.53 (± 0.36)22.2224.12 (± 0.54)1.906.7288.211.02 (± 0.45)22.3027.62 (± 0.42)5.3218.5293.29.82 (± 0.32)22.4129.84 (± 0.38)7.4325.3298.27.24 (± 0.27)22.0331.94 (± 0.56)9.9133.2303.25.05 (± 0.43)21.5035.17 (± 0.72)13.6745.1WBA II283.2NBNBNBNBNB298.2NBNBNBNBNBThe stoichiometries of 4-methoxygalactose binding to jacalin and WBA I were 4.01 ± 0.02 and 2.04 ± 0.09, respectively. The heat capacity (ΔCp) of 4-methoxygalactose binding to jacalin and WBA I were −345 ± 31 and −550 ± 22 J mol−1K−1, respectively (Fig. 1 G). NB, non-binding. Open table in a new tab The temperature dependence of the enthalpy (ΔH 0 b) (Fig. 1 G) and entropy (ΔS 0 b) (Fig. 1 H) was linear for the binding of 4-methoxygalactose to both jacalin and WBA I. There is almost no temperature dependence of the binding free energy (i.e. ΔΔG 0 b ≅ 0) (Fig. 1 I) within the temperature range examined because of significant enthalpy-entropy compensation (EEC) (Fig. 1 J). EEC apparently masks the differences in binding thermodynamics (14Swaminathan C.P. Surolia N. Surolia A. J. Am. Chem. Soc. 1998; 120: 5153-5159Crossref Scopus (85) Google Scholar,19Cooper A. Curr. Opin. Chem. Biol. 1999; 3: 557-563Crossref PubMed Scopus (185) Google Scholar). A close examination of the EEC plot (Fig. 1 J) reveals that the effect of EEC, in terms of the slope of the linear plots, is nearly the same for 4-methoxygalactose binding both to jacalin and WBA I. The y intercept (i.e. a position in the EEC plot where −TΔS 0 b is zero) of the EEC plot provides a measure of the condition at which all contributions from the enthalpic components proceed entirely to the free energy of the system, without any net change in entropic losses or gains. Thus, for enthalpically driven systems, such as the binding of 4-methoxygalactose to jacalin and WBA I, the differences iny intercepts of the respective EEC plots (25.2 ± 0.4 kJ mol−1 for jacalin and 22.4 ± 0.2 kJ mol−1 for WBA I) suggest different net contributions to the binding free energy emerging only in the form of different enthalpic components.In 400 MHz 1H-NMR experiments, the addition of WBA I or jacalin, but not WBA II, to a solution of 4-methoxygalactose led to broadening of the sugar resonances (Fig.2, A–C). The broadening was due to the slow exchange (τ m ≫T 2 m) of the sugar between free and lectin-bound states as attested to by a pronounced increase in line width with increase in temperature. Hence the line broadening effects are governed by the residence time of the anomers at the binding site. Analyses of the dependence of the net change in reciprocal line broadening as a function of 4-methoxygalactose concentration yielded K b values of 7.4 × 103 and 7.3 × 103 for the binding of α- and β-anomers, respectively, of 4-methoxygalactose to WBA I, whereas the K b for the jacalin−4-methoxygalactose complex was 1.5 × 104(Fig. 2 D). These values are in the range of titration calorimetric data (Table I). The ITC and NMR results together demonstrate unequivocally that the C-4 locus of galactose can tolerate substitution and yet not disrupt the specific carbohydrate binding ability of jacalin and WBA I, thus throwing light on the plasticity of their primary combining sites. Hence the binding site should be sufficiently flexible to accommodate this plasticity, which permits a promiscuous recognition of both galactose and 4-methoxygalactose.Figure 2400 MHz 1H-NMR partial spectra at 300 K of 4-methoxygalactose in the absence (A) and presence of WBA I (B) or jacalin (C). The spectra (——) in A andB were deconvoluted into two peaks each (- - -, – – –) corresponding to the α- and β-anomers of 4-methoxygalactose, whereas the spectra (——) in C did not deconvolute and represent the sum of α- and β-anomers.D, linear regression analysis of a plot (——) of the dependence of the reciprocal line broadening on 4-methoxygalactose concentration yielded K b values of 7.4 × 103 and 7.3 × 103 for the binding of α-(■) and β-(▵) anomers of 4-methoxygalactose, respectively, to WBA I, and 1.5 × 104 for the overall binding of 4-methoxygalactose (▪) to jacalin. The dissociation rate constants k −1 at 300 K derived fromD were 5.2 (± 0.2) s−1 and 5.1 (± 0.2) s−1 for the binding of α- and β-anomers of 4-methoxygalactose, respectively, to WBA I, and 30.5 (± 0.3) s−1 for the overall binding of 4-methoxygalactose to jacalin. The slopes and y intercepts, respectively, of the lines in D were 1.43 (r= 0.998) and 0.194 for (■), 1.42 (r = 0.998) and 0.196 for (▵), and 0.49 (r = 0.991) and 0.033 for (▪).View Large Image Figure ViewerDownload (PPT)These studies point to the existence of a C−H···O hydrogen bond between the 4-methoxy group of 4-methoxygalactose and the corresponding loci in the binding site of jacalin and WBA I. The presence of oxygen atoms in a large majority of molecules raises the possibility that the C−H···O hydrogen bond is widespread though not identified in many cases (20Taylor R. Kennard O. J. Am. Chem. Soc. 1982; 104: 5063-5070Crossref Scopus (2079) Google Scholar, 21Desiraju G.R. Acc. Chem. Res. 1996; 29: 441-449Crossref PubMed Scopus (1775) Google Scholar, 22Derewenda Z.S. Lee L. Derewenda U. J. Mol. Biol. 1995; 252: 248-262Crossref PubMed Scopus (497) Google Scholar). However, x-ray and neutron diffraction studies have shown that crystals of various organic molecules and biomolecules exhibit close C−H···X contacts (where X is an electronegative atom, in most cases oxygen) that show all the stereochemical hallmarks of hydrogen-bonds (21Desiraju G.R. Acc. Chem. Res. 1996; 29: 441-449Crossref PubMed Scopus (1775) Google Scholar). Recently, C−H···O interactions in collagen triple helix (23Bella J. Berman H.M. J. Mol. Biol. 1996; 264: 734-742Crossref PubMed Scopus (196) Google Scholar), DNA−protein complex (24Mandel-Gutfreund Y. Margalit H. Jernigan R.L. Zhurkin V.B. J. Mol. Biol. 1998; 277: 1129-1140Crossref PubMed Scopus (160) Google Scholar), thrombin-inhibitor complex (25Engh R.A. Brandstetter H. Sucher G. Eichinger A. Baumann U. Bode W. Huber R. Poll T. Rudolph R. von der Saal W. Structure. 1996; 4: 1353-1362Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), trypanothione reductase-trypanothione disulfide complex (26Bond C.S. Zhang Y. Berriman M. Cunningham M.L. Fairlamb A.H. Hunter W.N. Structure Fold. Des. 1999; 7: 81-89Abstract Full Text Full Text PDF Scopus (190) Google Scholar), active sites of serine hydrolases (27Derewenda Z.S. Derewenda U. Kobos P.M. J. Mol. Biol. 1994; 241: 83-93Crossref PubMed Scopus (203) Google Scholar), and helices involving proline residues (28Chakrabarti P. Chakrabarti S. J. Mol. Biol. 1998; 284: 867-873Crossref PubMed Scopus (169) Google Scholar) have also been identified. Cytosine-rich intercalated DNA quadruplexes not only contain intra-cytidine C−H···O hydrogen bonds but also display a systematic intermolecular C−H···O hydrogen bonding network between the deoxyribose sugar moieties of antiparallel backbones in the four-stranded molecule (29Berger I. Egli M. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12116-12121Crossref PubMed Scopus (153) Google Scholar). More recently, C−H···O hydrogen bonds have been found in the minor grooves of A-tracts in DNA double helices (30Ghosh A. Bansal M. J. Mol. Biol. 1999; 294: 1149-1158Crossref PubMed Scopus (73) Google Scholar), and, as a caveat, in cubanecarboxylic acids it is C−H···O bonds that determine O−H···O networks (31Kuduva S.S. Craig D.C. Nangia A. Desiraju G.R. J. Am. Chem. Soc. 1999; 121: 1936-1944Crossref Scopus (251) Google Scholar).There is ample evidence that “hydrogen-bonds” are composite, multi-center interactions that span a wide range of geometry and energy, with large chemical variations among the donor (X−H) and acceptor groups (21Desiraju G.R. Acc. Chem. Res. 1996; 29: 441-449Crossref PubMed Scopus (1775) Google Scholar). The angular distributions of C−H···O interactions for different types of C−H groups show that the directionality decreases with decreasing C−H polarization but is still clearly recognizable for methyl groups; on the other hand, for C−H···H−C van der Waals contacts, the isotropic angular characteristics are observed (32Steiner, T., and Desiraju, G. R. (1998) J. Chem. Soc. Chem. Commun. 891–892Google Scholar). There are many cases of multiple approaches by C−H groups in some donor-rich systems, with as many as four C−H groups forming contacts with a carbonyl oxygen atom (33Desiraju G.R. Kashino S. Coombs M.M. Glusker J.P. Acta Crystallogr. Sect. B Struct. Crystallogr. 1993; 49: 880-892Crossref PubMed Scopus (50) Google Scholar). Based on studies on simple alkynes and alkenes, it was found that the more acidic a particular type of C−H group, the shorter are the C−H···O bonds it forms (21Desiraju G.R. Acc. Chem. Res. 1996; 29: 441-449Crossref PubMed Scopus (1775) Google Scholar). It is interesting to note that the strength of a hydrogen bond in C≡C−H···O is close to 4–5 kcal mol−1, rivalling the O−H···O hydrogen bond in water (21Desiraju G.R. Acc. Chem. Res. 1996; 29: 441-449Crossref PubMed Scopus (1775) Google Scholar). The C-4 hydroxyl group serves as a donor of bidentate and tridentate hydrogen bonds in the complexes of WBA I−methyl-α-galactose (Ref. 9Prabu, M. M., Sankaranarayanan, R., Puri, K. D., Sharma, V., Surolia, A. (1998) J. Mol. Biol. 76, Vijayan, M., and Suguna, K. 2, 787–796Google Scholar and Fig.3 A) and jacalin−methyl-α-galactose (Ref. 8Sankaranarayanan R. Sekar K. Banerjee R. Sharma V. Surolia A. Vijayan M. Nat. Struct. Biol. 1996; 3: 596-603Crossref PubMed Scopus (225) Google Scholar and Fig. 3 C), respectively. As neither 4-deoxygalactose nor 4-fluorodeoxygalactose binds to either WBA I (10Swaminathan C.P. Gupta D. Sharma V. Surolia A. Biochemistry. 1997; 36: 13428-13434Crossref PubMed Scopus (23) Google Scholar) or to jacalin,3 the C-4 hydroxyl group contributes significantly to the reaction, predominantly as a hydrogen bond donor. Apparently the methoxy group at the C-4 locus of 4-methoxygalactose is able to fulfill such a role. This is consistent with the relatively greater acidity of a methyl group covalently bound to an oxygen atom than of that bound to an aliphatic carbon atom. These results thus substantiate the existence of C−H···O hydrogen bond(s) in the vicinity of the specificity-determining C-4 loci of the bound saccharide in the complexes of WBA I−4-methoxygalactose (Fig.3 B) and jacalin−4-methoxygalactose (Fig. 3 D). These C−H···O hydrogen bonding interactions involved in the recognition of 4-methoxygalactose by WBA I and jacalin appear to be stronger in magnitude (Table I) than those involved in the binding of galactose with WBA I (10Swaminathan C.P. Gupta D. Sharma V. Surolia A. Biochemistry. 1997; 36: 13428-13434Crossref PubMed Scopus (23) Google Scholar) and jacalin.3 The examples of 4-methoxygalactose−WBA I/jacalin interactions thus suggest that C−H···O hydrogen bonds in lectin−sugar interactions could contribute at least as significantly to the binding reaction as other hydrogen bonds.Figure 3Schematic representation of the primary combining site of lectins based on crystal structure and molecular modeling. WBA I complexed with methyl-α-galactose (A) (9Prabu, M. M., Sankaranarayanan, R., Puri, K. D., Sharma, V., Surolia, A. (1998) J. Mol. Biol. 76, Vijayan, M., and Suguna, K. 2, 787–796Google Scholar) and 4-methoxygalactose (B). Jacalin complexed with methyl-α-galactose (C) (8Sankaranarayanan R. Sekar K. Banerjee R. Sharma V. Surolia A. Vijayan M. Nat. Struct. Biol. 1996; 3: 596-603Crossref PubMed Scopus (225) Google Scholar) and 4-methoxygalactose (D). The amino acid residues tethering the specificity-determining C-4 locus of the saccharide are represented inboxes. Hydrogen bonds are represented as dotted lines.View Large Image Figure ViewerDownload (PPT)Aside from the main chain carbonyl of Thr-210 in WBA I at a distance of 4.39 Å from the methyl group oxygen at the C-4 locus of the modeled 4-methoxy-α-d-galactopyranoside, no atom other than the cognate binding site residues was present within 5.5 Å in the primary binding sites of WBA I and jacalin (11Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (36841) Google Scholar). The absence of nonpolar residues around the C-4 loci of methyl-α-galactose-bound complexes of WBA I and jacalin provide grounds to believe that the interaction of the 4-methoxy group of 4-methoxygalactose with the binding sites of WBA I and jacalin is not of a nonpolar nature. This is also supported by the observation that these binding reactions are predominantly enthalpically driven (Table I). Moreover, the root mean square deviation of C-α atoms of residues around the binding sites of native WBA I compared with those of WBA I complexed with methyl-α-galactose is less than 0.5 Å, suggesting the absence of significant conformational changes upon sugar binding.In conclusion, we have presented here for the first time evidence for the existence in lectin−carbohydrate recognition of C−H···O hydrogen bond(s) in the vicinity of the specificity-determining C-4 locus of the saccharide 4-methoxygalactose and the lectins WBA I and jacalin. The recognition of sugar molecules by carbohydrate-specific proteins, lectins, involves the establishment of an organized set of interactions within the binding site. Hydrogen-bonding interactions are one of the most important factors of molecular recognition in lectin−sugar interactions, along with van der Waals forces, which, although rather weak (often contributing only a fraction of 1 kcal mol−1 for each pair of atoms), are frequently numerous and together make a significant contribution to binding (1Goldstein I.J. Hayes C.E. Adv. Carbohydr. Chem. Biochem. 1978; 35: 127-340Crossref PubMed Scopus (1343) Google Scholar,2Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar). Despite the differences in the lectin folds and their modes of sugar binding, the specificity for the recognition of galactose is determined by interactions involving the C-4 locus of the saccharide (1Goldstein I.J. Hayes C.E. Adv. Carbohydr. Chem. Biochem. 1978; 35: 127-340Crossref PubMed Scopus (1343) Google Scholar, 2Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar, 3Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (188) Google Scholar, 4Elgavish S. Shaanan B. Trends Biochem. Sci. 1997; 22: 462-467Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 5Vijayan M. Chandra N. Curr. Opin. Struct. Biol. 1999; 9: 707-714Crossref PubMed Scopus (229) Google Scholar). Stereochemical evidence is emerging for a distinct sugar binding specificity-dependent distribution of hydrogen bond donors vis à vis the acceptors in the combining site of lectins (4Elgavish S. Shaanan B. Trends Biochem. Sci. 1997; 22: 462-467Abstract Full Text PDF PubMed Scopus (218) Google Scholar). The C-4 locus of the monosaccharide within the primary binding site of galactose/N-acetylgalactosamine-specific lectins has hitherto been considered to be absolutely invariant. Though belonging to different families, jacalin, a Moraceaemember, and WBA1 I, aLeguminosae member, both are galactose/N-acetylgalactosamine-specific lectins (6Sastry M.V. Banarjee P. Patanjali S.R. Swamy M.J. Swarnalatha G.V. Surolia A. J. Biol. Chem. 1986; 261: 11726-11733Abstract Full Text PDF PubMed Google Scholar, 7Khan M.I. Sastry M.V. Surolia A. J. Biol. Chem. 1986; 261: 3013-3019Abstract Full Text PDF PubMed Google Scholar). Whereas jacalin displays a β-prism tertiary structural fold wherein a unique post-translationally generated N-terminal glycine residue serves as a critical determinant of galactose specificity (8Sankaranarayanan R. Sekar K. Banerjee R. Sharma V. Surolia A. Vijayan M. Nat. Struct. Biol. 1996; 3: 596-603Crossref PubMed Scopus (225) Google Scholar), WBA I contains a legume lectin fold (9Prabu, M. M., Sankaranarayanan, R., Puri, K. D., Sharma, V., Surolia, A. (1998) J. Mol. Biol. 76, Vijayan, M., and Suguna, K. 2, 787–796Google Scholar). During the course of mapping and establishing the hydrogen bond donor-acceptor relationship of the primary combining site of galactose-specific lectins (10Swaminathan C.P. Gupta D. Sharma V. Surolia A. Biochemistry. 1997; 36: 13428-13434Crossref PubMed Scopus (23) Google Scholar), unexpectedly, we have discovered that the 4-methoxy derivative ofd-galactopyranoside (4-methoxygalactose) binds, with affinities comparable with that of methyl-α-galactose, to theMoraceae lectin jacalin and the Leguminosaelectin WBA I but not to the related WBA II. 2Srinivas, V. R., Acharya, S., Rawat, S., Sharma, V., and Surolia, A. (2000) FEBS Lett. 474, 76–82.2Srinivas, V. R., Acharya, S., Rawat, S., Sharma, V., and Surolia, A. (2000) FEBS Lett. 474, 76–82. RESULTS AND DISCUSSIONThe ITC experiments directly detected the evolution of heats of binding when fixed aliquots of 4-methoxygalactose solution were added into either jacalin solution (Fig.1 A) or WBA I solution (Fig.1 C). These data, analyzed by iterated non-linear least squares fitting procedures, were found to best fit the simplest identical site model indicating that both the lectins bind to 4-methoxygalactose (Fig. 1, B and D). The reactions were driven by dominant favorable enthalpic contributions (Table I). These results, contrary to the usual expectation of the indispensability of the unsubstituted C-4 hydroxyl of the saccharide, clearly demonstrate the capability of 4-methoxygalactose to bind to both WBA I as well as jacalin. This reflects the existence of 4-methoxygalactose binding ability in galactose/N-acetylgalactosamine-specific lectin members from at least two unrelated families. To test whether 4-methoxygalactose displays a promiscuous binding to other lectins with similar monosaccharide specificity, the binding of 4-methoxygalactose to WBA II, a dimeric acidic lectin sequentially and structurally similar to WBA I, was tested. The results of such an ITC experiment indicate no binding of 4-methoxygalactose to WBA II (Fig. 1, E andF). That the non-binding of 4-methoxygalactose to WBA II was not due to loss of activity of WBA II was confirmed by testing the binding of 2′fucosyllactose to WBA II; the thermodynamic parameters obtained, K b = 1.0 × 105 and ΔH b 0 = 43.2 kJ mol−1 at 298.2 K, agreed very well with previously published values (12Srinivas V.R. Singha N.C. Schwarz F.P. Surolia A. Carbohydr. Lett. 1998; 3: 129-136Google Scholar). In contrast, none of these galactose/N-acetylgalactosamine-specific lectins, WBA I, WBA II, or jacalin, bound 4-methoxyglucose. The inherent inability of WBA II to recognize 4-methoxygalactose is perhaps due to dominant steric factors around its specificity-determining C-4 locus. The ability of 4-methoxygalactose to bind to WBA I as well as jacalin, together with its inability to bind to WBA II, is consistent with the formation of favorable interactions from the vicinity of the C-4 locus of 4-methoxygalactose with the corresponding binding site residues in WBA I and jacalin. It appears that the disposition of amino acid residues in the vicinity of the C-4 locus of 4-methoxygalactose within the binding sites of WBA I and jacalin permits the efficient binding of 4-methoxygalactose by WBA I and" @default.
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