Thermodynamics of Lectin-Carbohydrate Interactions
1997; Elsevier BV; Volume: 272; Issue: 10 Linguagem: Inglês
10.1074/jbc.272.10.6388
ISSN1083-351X
AutoresDipti Gupta, Tarun K. Dam, Stefan Oscarson, C. Fred Brewer,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe trisaccharide 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was previously shown by titration microcalorimetry to bind to the lectin concanavalin A (ConA) with nearly −6 kcal mol−1 greater enthalpy change and 60-fold higher affinity than methyl-α-D-mannopyranoside (Mandal, D. K., Kishore, N., and Brewer, C. F. (1994) Biochemistry 33, 1149-1156). Similar studies of the binding of a series of monodeoxy derivatives of the α(1-3) residue of the trimannoside showed that this arm was required for high affinity binding (Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., III, Oscarson, S., and Brewer, C. F. (1994) Biochemistry 33, 1157-1162). In the present paper, a series of monodeoxy derivatives of the α(1-6) arm and "core" Man residue of the trimannoside as well as dideoxy and trideoxy analogs were synthesized. Isothermal titration microcalorimetry experiments establish that the 3-, 4-, and 6-hydroxyl groups of the α(1-6)Man residue of the trimannoside binds to the lectin, along with the 2- and 4-hydroxyl groups of the core Man residue and the 3- and 4-hydroxyl groups of the α(1-3)Man residue. Dideoxy analogs and trideoxy analogs showed losses of affinities and enthalpy values consistent with losses in binding of specific hydroxyl groups of the trimannoside. The free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside determined from the corresponding monodeoxy analogs are observed to be nonlinear, indicating differential contributions of the solvent and protein to the thermodynamics of binding of the analogs. The thermodynamic solution data agree well with the recent x-ray crystal structure of ConA complexed with the trimannoside (Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976). The trisaccharide 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was previously shown by titration microcalorimetry to bind to the lectin concanavalin A (ConA) with nearly −6 kcal mol−1 greater enthalpy change and 60-fold higher affinity than methyl-α-D-mannopyranoside (Mandal, D. K., Kishore, N., and Brewer, C. F. (1994) Biochemistry 33, 1149-1156). Similar studies of the binding of a series of monodeoxy derivatives of the α(1-3) residue of the trimannoside showed that this arm was required for high affinity binding (Mandal, D. K., Bhattacharyya, L., Koenig, S. H., Brown, R. D., III, Oscarson, S., and Brewer, C. F. (1994) Biochemistry 33, 1157-1162). In the present paper, a series of monodeoxy derivatives of the α(1-6) arm and "core" Man residue of the trimannoside as well as dideoxy and trideoxy analogs were synthesized. Isothermal titration microcalorimetry experiments establish that the 3-, 4-, and 6-hydroxyl groups of the α(1-6)Man residue of the trimannoside binds to the lectin, along with the 2- and 4-hydroxyl groups of the core Man residue and the 3- and 4-hydroxyl groups of the α(1-3)Man residue. Dideoxy analogs and trideoxy analogs showed losses of affinities and enthalpy values consistent with losses in binding of specific hydroxyl groups of the trimannoside. The free energy and enthalpy contributions to binding of individual hydroxyl groups of the trimannoside determined from the corresponding monodeoxy analogs are observed to be nonlinear, indicating differential contributions of the solvent and protein to the thermodynamics of binding of the analogs. The thermodynamic solution data agree well with the recent x-ray crystal structure of ConA complexed with the trimannoside (Naismith, J. H., and Field, R. A. (1996) J. Biol. Chem. 271, 972-976). INTRODUCTIONThe ability of concanavalin A (ConA) 1The abbreviations used are: ConAconcanavalin A, lectin from jack beanN-linkedasparagine-linkedMe-α-Manmethyl-α-D-mannopyranoside1methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside2methyl-6-O-(α-D-mannopyranosyl)-3-O-(2-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside3methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside4methyl-6-O-(α-D-mannopyranosyl)-3-O-(4-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside5methyl-6-O-(α-D-mannopyranosyl)-3-O-(6-deoxy-α-Dmannopyranosyl)-α-D-mannopyranoside6methyl-6-O-(2-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside7methyl-6-O-(3-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside8methyl-6-O-(4-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside9methyl-6-O-(6deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside10methyl-6-O-(α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-2-deoxy-α-D-mannopyranoside11methyl-6-O-(α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-4-deoxy-α-D-mannopyranoside12methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-2-deoxy-α-D-mannopyranoside13methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-4-deoxy-α-D-mannopyranoside14methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)2,4-dideoxy-α-D-mannopyranoside. to bind with high affinity to certain N-linked carbohydrates has made it a valuable tool to investigate the carbohydrates of normal and transformed cells, as well as to isolate carbohydrates, glycoconjugates, and cells on ConA-affinity matrixes (1Bittiger H. Schnebli H.P. Concanavalin A as a Tool. John Wiley and Sons, New York1976Google Scholar, 2Lis H. Sharon N. Biochem. Plants. 1981; 6: 371-447Google Scholar). Thus, it is important to establish the nature of the molecular interactions between N-linked carbohydrates and ConA in order to understand the specificity of the lectin for cellular carbohydrates.ConA is a tetramer above pH 7 and a dimer below pH 6, with each monomer (Mr = 25, 600) possessing one saccharide-binding site as well as a transition metal ion site (S1) (typically Mn2+) and a Ca2+ site (S2) (3Kalb A.J. Levitzki A. Biochem. J. 1968; 109: 669-672Crossref PubMed Scopus (214) Google Scholar, 4Yariv J. Kalb A.J. Levitzki A. Biochim. Biophys. Acta. 1968; 165: 303-305Crossref PubMed Scopus (186) Google Scholar, 5Brewer C.F. Brown R.D.I. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (59) Google Scholar). The three-dimensional structure of the lectin at 1.75-Å resolution has been determined by x-ray diffraction analysis (6Hardman K.D. Agarwal R.C. Freiser M.J. J. Mol. Biol. 1982; 157: 69-86Crossref PubMed Scopus (157) Google Scholar), and a complex with Me-α-Man to 2.9-Å resolution (7Derewenda Z. Yariv J. Helliwell J.R. Kalb A.J. Dodson E.J. Papiz M.Z. Wan T. Campbell J. EMBO J. 1989; 8: 2189-2193Crossref PubMed Scopus (267) Google Scholar). Recently, the x-ray crystal structure of ConA bound with methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside to 2.3 Å has been reported (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Early studies by Goldstein and co-workers (9Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins. Academic Press, Inc., New York1986: 35-244Google Scholar) established that ConA has specificity for α-pyranose forms of Glc and Man, which contain similar hydroxyl group configurations at the 3-, 4-, and 6-positions. Monodeoxy derivatives of the monosaccharides at these positions showed essentially complete loss of binding to the lectin, thus establishing the specificity of the so-called "monosaccharide-binding site" in ConA. Later studies demonstrated that certain N-linked oligomannose and complex-type oligosaccharides possessed much higher affinities (∼50-fold or greater) than the monosaccharide methyl-α-D-mannopyranoside (Me-α-Man) (10Bhattacharyya L. Brewer C.F. Eur. J. Biochem. 1989; 178: 721-726Crossref PubMed Scopus (47) Google Scholar), thus suggesting extended site binding interactions with the lectin. The trisaccharide moiety 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is part of all N-linked oligosaccharides, was shown to bind with nearly 100-fold higher affinity than Me-α-Man, and to induce conformational changes in ConA similar to those of the larger N-linked carbohydrates (10Bhattacharyya L. Brewer C.F. Eur. J. Biochem. 1989; 178: 721-726Crossref PubMed Scopus (47) Google Scholar, 11Brewer C.F. Bhattacharyya L. J. Biol. Chem. 1986; 261: 7306-7310Abstract Full Text PDF PubMed Google Scholar). These results indicated that the trimannosyl moiety in N-linked carbohydrates was primarily responsible for their high affinity binding to ConA.Detailed insights into the specificity of carbohydrate-protein interactions requires not only relative binding affinity data, but also thermodynamic data to establish whether extended binding site interactions occur. We recently described titration microcalorimetry studies of the binding of methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside (1) (Fig. 1) to ConA (12Mandal D.K. Kishore N. Brewer C.F. Biochemistry. 1994; 33: 1149-1156Crossref PubMed Scopus (262) Google Scholar). The results showed that 1 possesses nearly 6 kcal mol−1 greater change in binding enthalpy (−ΔH) than Me-α-Man, thus providing direct evidence for extended recognition site by the lectin of the trimannoside epitope. Similar studies with deoxy analogs of the α(1-3)Man residue of the trimannoside established that the 3-OH on this arm is required for high affinity binding (13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar).In the present study a series of monodeoxy derivatives of the α(1-6) arm and "core" Man residue of the trimannoside, as well as dideoxy and trideoxy analogs of 1 (Fig. 1), were synthesized and their binding to ConA investigated by isothermal titration microcalorimetry measurements. In light of the recently reported x-ray crystal structure of ConA and the trimannoside (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), the present results provide information on the energetics of binding of the trimannoside to ConA, and, in turn, on the structure of the complex in solution.RESULTS AND DISCUSSIONOur previous titration calorimetry studies showed that ConA binds to trimannoside 1 which is present in all N-linked carbohydrates with nearly −6 kcal mol−1 greater enthalpy change (−ΔH) and 60-fold higher affinity than Me-α-Man (Table I) (12Mandal D.K. Kishore N. Brewer C.F. Biochemistry. 1994; 33: 1149-1156Crossref PubMed Scopus (262) Google Scholar). These results indicate that the high affinity of the the trimannoside moiety is due to extended site interactions. In order to examine the nature of these extended site interactions, a complete set of monodeoxy analogs of 1 as well as two dideoxy and a trideoxy analog were synthesized and their thermodynamics of binding determined by titration microcalorimetry. The results have provided structural information on the complex in solution which has been compared with the recently reported x-ray crystal structure of the complex (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Table I.Thermodynamic parameters derived from the titration of ConA at pH 7.2 with saccharides at 27°CCarbohydrate (abbr.)CarbohydrateLectinKaaErrors in Ka values were between 2 and 10%.−ΔGΔΔG−ΔHbErrors in ΔH and TΔS were ±0.1 to ±0.2.ΔΔ(H)cRelative to 1.−TΔSbErrors in ΔH and TΔS were ±0.1 to ±0.2.mMM−1× 10−4kcal mol−1MeαMan46.00.480.82dData taken from Mandal et al (13) and included here for comparison.5.32.58.26.22.91 "Trimannoside"7.00.1349.0dData taken from Mandal et al (13) and included here for comparison.7.814.46.62 α(1-3)2-deoxy6.20.1456.8dData taken from Mandal et al (13) and included here for comparison.7.9−0.114.5−0.16.63 α(1-3)3-deoxy9.00.145.39dData taken from Mandal et al (13) and included here for comparison.6.51.411.03.44.54 α(1-3)4-deoxy9.00.249.26.81.012.32.15.55 α(1-3)6-deoxy6.40.1539.6dData taken from Mandal et al (13) and included here for comparison.7.7−0.114.00.46.36 α(1-6)2-deoxy6.20.2350.17.80.014.9−0.57.17 α(1-6)3-deoxy14.50.333.886.31.511.23.24.98 α(1-6)4-deoxy7.00.232.546.01.811.72.75.79 α(1-6)6-deoxy12.50.333.016.11.711.62.85.510 "core"2-deoxy12.50.2411.76.90.913.41.06.511 "core"4-deoxy9.30.264.636.41.412.12.35.712 α(1-3)3-deoxy, core 2-deoxy42.50.543.606.21.610.63.84.413 α(1-3)3-deoxy, core 4-deoxy7.00.241.935.91.99.74.73.814 α(1-3)3-deoxy, core 2,4-deoxy45.00.750.995.52.38.75.73.2a Errors in Ka values were between 2 and 10%.b Errors in ΔH and TΔS were ±0.1 to ±0.2.c Relative to 1.d Data taken from Mandal et al (13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar) and included here for comparison. Open table in a new tab Binding of Monodeoxy Analogs of Trimannoside 1Our previous studies examined the thermodynamics of binding of a series of monodeoxy analogs of 1 possessing substitutions on the α(1-3) arm (13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar). The results, which are shown in Table I for comparison, indicate that the 2- (2) and 6-deoxy (5) analogs (Fig. 1) possess similar affinities, −ΔH and entropy (TΔS) values as that of the parent trimannoside. However, the 3-deoxy analog (3) showed a nearly 10-fold decrease in affinity and a 3.4 kcal mol−1 decrease in −ΔH with respect to 1, indicating its involvement in binding. Although we previously reported no change in the thermodynamics of binding of the 4-deoxy analog (4) relative to 1, a reinvestigation shows that 4 binds to ConA with a 5-fold decrease in affinity and a 2.1 kcal mol−1 decrease in −ΔH with respect to 1 (Table I). Thus, the 3- and 4-hydroxyl groups on the α(1-3) arm of 1 bind to ConA.Our previous studies also revealed that analogs of 1 with a Glc or Gal residue substituted on the α(1-6) arm possessed decreased affinities and −ΔH values relative to 1 (13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar). These results provided evidence that the α(1-6)Man residue binds to the so-called "monosaccharide"-binding site of ConA, by analogy to the requirements of monosaccharide binding to ConA (9Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins. Academic Press, Inc., New York1986: 35-244Google Scholar). However, in order to directly determine the interactions of the α(1-6) arm of 1 with ConA, the 2- (6), 3- (7), 4- (8), and 6-deoxy (9) derivatives of the α(1-6)Man arm of 1 were synthesized. The thermodynamic binding data for these derivatives are shown in Table I. The binding parameters of 6 are nearly the same as that of 1, indicating no binding of the the 2-OH group of the α(1-6) arm. On the other hand, the Ka and ΔH values for 7, 8, and 9 are significantly lower than that of 1, indicating the involvement of 3-, 4-, and 6-OH of the α(1-6)Man residue of the trimannoside in binding (19Wells T.N.C. Fersht A.R. Biochemistry. 1986; 25: 1881-1886Crossref PubMed Scopus (112) Google Scholar). The magnitude of the reductions in Ka and ΔH for 7-9 are similar to those observed for 3 and 4 above (Table I). Since the 3-, 4-, and 6-OH groups of the monosaccharides Man and Glc are required for binding to ConA (9Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins. Academic Press, Inc., New York1986: 35-244Google Scholar), the data are consistent with binding of the α(1-6)Man of 1 to the "monosaccharide site" of the lectin.Titration microcalorimetry data for the 2-deoxy (10) and 4-deoxy (11) derivatives of the central Man residue of 1 are shown in Table I. The results show reductions in Ka and −ΔH for both analogs, indicating the involvement of the 2- and 4-OH of the central Man residue of 1 in binding to ConA.Binding of Dideoxy and Trideoxy Analogs of 1The 2,3- (12) and 4,3- (13) dideoxy analogs of 1, and trideoxy analog 14 (Fig. 1) were also synthesized. Table I shows that the 12 binds about 14-fold more weakly than 1 and possesses a ΔH of −10.6 kcal mol−1 which is a loss of −3.8 kcal mol−1 compared to 1. This reflects the combined loss in ΔH by the 2-deoxy analog 10 and 3-deoxy derivative 3 relative to 1. Table I shows that 13 binds about 25-fold more weakly than 1, and possesses a ΔH of −9.7 kcal mol−1 which is −4.7 kcal mol−1 less favorable than that of 1. This reflects the combined loss in ΔH of the 3-deoxy derivative 3 and 4-deoxy derivative 11 relative to 1.Trideoxy analog 14, which possesses deoxy substitutions at the 3-OH group on the α(1-3) arm and at the 2- and 4-OH groups of the central Man residue of 1, exhibits a Ka value about 50-fold lower than that of 1 (Table I). The ΔH of 14 is −8.7 kcal mol−1 which is 5.7 kcal mol−1 less than that of 1. This reflects losses in ΔH of the corresponding monodeoxy analogs 3, 10, and 11, relative to 1.Comparison with the X-ray Crystal Structure of 1 Complexed to ConAThe x-ray structure of the complex formed by the free sugar of 1 with ConA has recently been reported (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). A view of the H-bonding interactions between 1 and the binding site of the lectin is shown in Fig. 3, with the individual hydrogen bonds and their distances listed in Table II. The crystal structure indicates that the α(1-6)Man residue of 1 binds via its 3-, 4-, and 6-hydroxyl groups in the same manner as Me-α-Man in its crystalline complex with the lectin (7Derewenda Z. Yariv J. Helliwell J.R. Kalb A.J. Dodson E.J. Papiz M.Z. Wan T. Campbell J. EMBO J. 1989; 8: 2189-2193Crossref PubMed Scopus (267) Google Scholar). These results agree with the thermodynamic data for the 7, 8, and 9 in Table I. The x-ray data also shows binding of the 3-OH of the α(1-3) Man residue to the N-H and side chain O of Thr-15, and the 4-OH of the α(1-3)Man residue to the side chain -OH of Thr-15 (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) (Fig. 3; Table II). These results agree with the thermodynamic data for 3 and 4 in Table I. The x-ray data also shows binding of the 2-OH of the central Man residue of 1 to a water molecule which, in turn, is bound to the protein, as shown in Fig. 3, and the 4-OH of the central Man residue to the aromatic 4-OH of Tyr-12 (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). These results agree with the thermodynamic data for 10 and 11 in Table I. Thus, the titration calorimetry data in Table I which provides structural information on the solution complex of 1 with ConA agrees with the x-ray structure of the complex (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Fig. 3View of the free sugar of trimannoside 1 (no α-anomeric methoxy group) in the binding site of ConA as determined by x-ray crystallography. This view emphasizes the hydrogen bonding interactions of the trimannoside with residues of the lectins. The trisaccharide is shown in stick format, with the central Man indicated by C, the α(1-6)Man by 6 and the α(1-3)Man by 3. The distances and assignments for these hydrogen bonds are given in Table II. The data are from Naismith and Field (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table II.Hydrogen bonding and polar contact ( 45°) from the linearity expected for a hydrogen bond.SugarProteinDistanceÅα(1-6)ManO-3Arg-228 N2.9O-4Asn-14ND22.9O-4Asp-208OD12.7O-4bOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.Arg-228 N3.5O-5Leu-99 N2.9O-6Asp-208OD22.9O-6Tyr-100 N3.1O-6bOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.Leu-993.1Central ManO-2OWbOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.2.6O-2bOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.Asp-16OD13.1O-4Tyr-12 OH2.8α(1-3)ManO-3Thr-15 N2.8O-3Thr-15OG12.9O-3bOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.Pro-13 O2.9O-4Thr-15OG13.1O-4bOW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228.Asp-16 N3.0a These contacts are within hydrogen bonding distance; however, the geometry of the donor-H-acceptor atoms differs substantially (>45°) from the linearity expected for a hydrogen bond.b OW is the structurally conserved water molecule. This is bound by protein residues Asn-14, Asp-16, and Arg-228. Open table in a new tab Nonlinearity of the ΔΔH and ΔΔG Values of the Individual Hydroxyl Groups of 1The present thermodynamic data indicate that the ΔΔH values for the monodeoxy analogs in Table I are nonlinear. For example, the sum of the ΔΔH values for monodeoxy analogs 7, 8, and 9 is ∼−10.5 kcal mol−1 (Table I). To a first approximation, this represents the combined ΔH contribution of the 3-, 4-, and 6-OH of the α(1-6)Man residue of 1 at the monosaccharide site of the lectin. This value can be compared to the ΔH for Me-α-Man binding of −8.2 kcal mol−1 to the same site. Furthermore, the value of −10.5 kcal mol−1 does not take into account the ΔH contribution of the ring oxygen of the α(1-6)Man residue of 1 (Fig. 3) which would make this value even greater and hence the difference with the ΔH of Me-α-Man larger. Similarly, the combined ΔΔH values for the 3-OH and 4-OH of the α(1-3)Man residue of 1 obtained from 3 and 4, respectively, and the 2-OH and 4-OH of the central Man residue obtained from 10 and 11, respectively, is ∼−8.8 kcal mol−1. This can be compared to the difference in ΔH between 1 and Me-α-Man of −6.2 kcal mol−1 which reflects binding of the α(1-3)Man and the central Man residues of 1. Furthermore, the sum of the ΔΔH values for the 3-, 4- and 6-OH of the α(1-6)Man residue (7, 8, and 9), the 3- and 4-OH of the α(1-3)Man residue (3 and 4), and the 2- and 4-OH of the central Man residue (10 and 11) is −19.3 kcal mol−1 which is greater than the ΔH for 1 of −14.4 kcal mol−1. Thus, the sum of the ΔΔH values for the hydroxyl groups of 1 obtained from the monodeoxy analogs in Table I does not correspond to the measured ΔH of 1. In all of the above cases, the sum of the ΔΔH values for specific hydroxyl groups on certain Man residues of 1 obtained from the corresponding monodeoxy analogs is greater than the measured ΔH for that residue(s).This nonlinear relationship in ΔΔH is also present in the di- and trideoxy analogs of 1. ΔΔH for dideoxy analog 12 is −3.8 kcal mol−1 as compared to the sum of the ΔΔH values for corresponding monodeoxy analogs 3 and 10 of −4.4 kcal mol−1. Likewise, ΔΔH for dideoxy analog 13 is −4.7 kcal mol−1 as compared to the sum of the ΔΔH values for corresponding monodeoxy analogs 3 and 11 of −5.7 kcal mol−1. In the case of trideoxy analog 14, its ΔΔH value is −5.7 kcal mol−1 as compared to −6.7 kcal mol−1 for 3, 10, and 11. Thus, the ΔΔH values for the monodeoxy analogs are nonlinear in terms of their contributions to the ΔΔH values for the two dideoxy analogs and the trideoxy analog.The same nonlinearity is also present in the ΔΔG values of the monodeoxy analogs. For example, the sum of the ΔΔG values for 3, 4, 10, and 11 is −4.7 kcal mol−1, however, the difference in ΔΔG between 1 and Me-α-Man is −2.5 kcal mol−1 (Table I). In addition, the sum of the ΔΔG values for 3 and 10 is −2.3 kcal mol−1 while the ΔΔG for 12, the corresponding dideoxy analog, is −1.6 kcal mol−1. The sum of the ΔΔG values for 3 and 11 is −2.8 kcal mol−1 while the ΔΔG for 13, the corresponding dideoxy analog, is −1.9 kcal mol−1. The sum of the ΔΔG values for 3, 10, and 11 is −3.7 kcal mol−1 while the ΔΔG value for 14, the corresponding trideoxy analog, is −2.3 kcal mol−1. Thus, the ΔΔG values for the monodeoxy analogs are nonlinear in terms of their contributions to the ΔΔG values for the two dideoxy analogs and the trideoxy analog.The ΔΔH and ΔΔG values for each monodeoxy analog of 1 also do not scale with the number of H-bonds at each position as determined from x-ray crystallography (Fig. 3; Table II). For example, ΔΔH for 7 is −3.2 kcal mol−1, as compared to −2.7 and −2.8 kcal mol−1 for 8 and 9, respectively. However, the x-ray crystal structure shows only one H-bond from the protein to the 3-OH of the α(1-6)Man residue and at least two strong H-bonds each to the 4-OH and 6-OH of the α(1-6)Man residue. Thus, the ΔΔH of −3.2 kcal mol−1 for 7 reflects the loss of a single H-bond with the protein, however, the ΔΔH values for 8 (−2.7 kcal mol−1) and 9 (−2.8 kcal mol−1) do not scale with the loss of two H-bonds. The type of H-bonds involved (Table II) does not provide an explanation for this apparent discrepancy. Likewise, the ΔΔH for 3 (−3.4 kcal mol−1) is nearly the same as that for 7 (−3.2 kcal mol−1), even though in the latter case there are two H-bonds to the 3-OH of the α(1-3)Man residue. Thus, the ΔΔH values for the monodeoxy analogs are not proportional to the number or type of H-bonds involved at specific hyroxyl groups of 1.This same lack of scaling is also present in the ΔΔG values (Table I). This is of particular interest since it has been suggested that the free energy associated with elimination of a H-bond between an uncharged donor/acceptor pair is 0.5-1.5 kcal mol−1, and between a neutral-charged pair 3.5-4.5 kcal mol−1 (20Fersht A.R. Shi J.-P. Knill-Jones J. Lowe D.M. Wilkinson A.J. Blow D.M. Brick P. Cartger P. Waye M.M.Y. Winter G. Nature. 1985; 314: 235-238Crossref PubMed Scopus (984) Google Scholar). The data in Table I, however, indicate no such relationship in the free energy difference (ΔΔG) of monodeoxy analogs that represent the loss of one or more H-bonds such as 7 versus 8 and 9.The presence of nonlinear relationships in the ΔΔH and ΔΔG values for the deoxy analogs in Table I indicates other contributions to these terms such as solvent and protein effects. Thus, the magnitude of the ΔΔH and ΔΔG values represent not only the loss of the H-bond(s) involved, but also differences in the solvent and protein contributions to binding of 1 and the deoxy analogs. Indeed, a recent study suggests a substantial contribution of solvent to the ΔH of binding of 1 to ConA (21Chervenak M.C. Toone E.J. J. Am. Chem. Soc. 1994; 116: 10533-10539Crossref Scopus (231) Google Scholar). Thus, titration microcalorimetry measurements of the binding of deoxy analogs of a substrate to a macromolecule do not provide direct measurements of the free energy and enthalpy of the H-bonding involved.Enthalpy-Entropy CompensationEnthalpy-entropy compensation plots have previously been observed for carbohydrate interactions with lectins (22Schwarz F.P. Puri K.D. Bhat R.G. Surolia A. J. Biol. Chem. 1993; 268: 7668-7677Abstract Full Text PDF PubMed Google Scholar, 23Lemieux R.U. Delbaere L.T.J. Beierbeck H. Spohr U. Ciba Found. Symp. 1991; 158: 231-248PubMed Google Scholar) and antibodies (24Herrons J.N. Kranz D.M. M. J.D. Voss E.W. Biochemistry. 1986; 25: 4602-4609Crossref PubMed Scopus (78) Google Scholar, 25Brummell D.A. Sharma V.P. Anand N.N. Bilous D. Dubuc G. Michniewicz J. MacKenzie C.R. Sadowska J. Sigurskjold B.W. Sinnott B. Young N.M. Bundle D.R. Narang S.A. Biochemistry. 1993; 32: 1180-1187Crossref PubMed Scopus (70) Google Scholar, 26Sigurskjold B.W. Bundle D.R. J. Biol. Chem. 1992; 267: 8371-8376Abstract Full Text PDF PubMed Google Scholar), and attributed to the unique properties of water (23Lemieux R.U. Delbaere L.T.J. Beierbeck H. Spohr U. Ciba Found. Symp. 1991; 158: 231-248PubMed Google Scholar). A plot of the −ΔH versus −TΔS values at 300 K for ConA binding to the oligosaccharides in Table I shows that it is also compensatory (Fig. 4). The plot shows a linear relationship with a slope of 1.55 and the correlation coefficient to 0.94. The enthalpy-entropy plot in Fig. 3 is similar to those reported earlier for other lectin-carbohydrate interactions in that their slopes are greater than unity (27Munske G.R. Krakauer H. Magnuson J.A. Arch. Biochem. Biophys. 1984; 233: 582-587Crossref PubMed Scopus (20) Google Scholar, 28Lemieux R.U. Chem. Soc. Rev. 1989; 18: 347-374Crossref Google Scholar), in contrast to antibody-carbohydrate interactions where the slope is often less than unity (25Brummell D.A. Sharma V.P. Anand N.N. Bilous D. Dubuc G. Michniewicz J. MacKenzie C.R. Sadowska J. Sigurskjold B.W. Sinnott B. Young N.M. Bundle D.R. Narang S.A. Biochemistry. 1993; 32: 1180-1187Crossref PubMed Scopus (70) Google Scholar, 26Sigurskjold B.W. Bundle D.R. J. Biol. Chem. 1992; 267: 8371-8376Abstract Full Text PDF PubMed Google Scholar). A slope greater than unity means that the free energy of binding is predominantly driven by enthalpy, while a slope less than unity indicates dominant entropy contributions.Fig. 4Plot of −ΔH versus −TΔS for the binding of ConA to 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, trisaccharide 1, monodeoxy derivatives 2-11, dideoxy derivatives 12-13, and trideoxy derivative 14 at 27°C (300 K). Thermodynamic values were obtained from Refs. 12Mandal D.K. Kishore N. Brewer C.F. Biochemistry. 1994; 33: 1149-1156Crossref PubMed Scopus (262) Google Scholar and 13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar and the present study.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SummaryThe present study provides a thermodynamic description of the binding of trimannoside 1 and a series of mono-, di-, and trideoxy analogs to ConA using titration microcalorimetry. The results are consistent with binding of the 3-, 4-, and 6-hydroxyls of the α(1-6)Man, the 2- and 4-OH groups of the core Man, and the 3- and 4-OH on the α(1-3) an of 1 to the lectin. These results agree with the recently described x-ray crystal structure of the complex (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Nonlinear effects in the ΔΔH and ΔΔG values of the deoxy analogs indicate differential contributions of the solvent and protein to their binding, and not direct measurements of the loss in H-bonding interactions. INTRODUCTIONThe ability of concanavalin A (ConA) 1The abbreviations used are: ConAconcanavalin A, lectin from jack beanN-linkedasparagine-linkedMe-α-Manmethyl-α-D-mannopyranoside1methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside2methyl-6-O-(α-D-mannopyranosyl)-3-O-(2-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside3methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside4methyl-6-O-(α-D-mannopyranosyl)-3-O-(4-deoxy-α-D-mannopyranosyl)-α-D-mannopyranoside5methyl-6-O-(α-D-mannopyranosyl)-3-O-(6-deoxy-α-Dmannopyranosyl)-α-D-mannopyranoside6methyl-6-O-(2-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside7methyl-6-O-(3-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside8methyl-6-O-(4-deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside9methyl-6-O-(6deoxy-α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside10methyl-6-O-(α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-2-deoxy-α-D-mannopyranoside11methyl-6-O-(α-D-mannopyranosyl)-3-O-(α-D-mannopyranosyl)-4-deoxy-α-D-mannopyranoside12methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-2-deoxy-α-D-mannopyranoside13methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)-4-deoxy-α-D-mannopyranoside14methyl-6-O-(α-D-mannopyranosyl)-3-O-(3-deoxy-α-D-mannopyranosyl)2,4-dideoxy-α-D-mannopyranoside. to bind with high affinity to certain N-linked carbohydrates has made it a valuable tool to investigate the carbohydrates of normal and transformed cells, as well as to isolate carbohydrates, glycoconjugates, and cells on ConA-affinity matrixes (1Bittiger H. Schnebli H.P. Concanavalin A as a Tool. John Wiley and Sons, New York1976Google Scholar, 2Lis H. Sharon N. Biochem. Plants. 1981; 6: 371-447Google Scholar). Thus, it is important to establish the nature of the molecular interactions between N-linked carbohydrates and ConA in order to understand the specificity of the lectin for cellular carbohydrates.ConA is a tetramer above pH 7 and a dimer below pH 6, with each monomer (Mr = 25, 600) possessing one saccharide-binding site as well as a transition metal ion site (S1) (typically Mn2+) and a Ca2+ site (S2) (3Kalb A.J. Levitzki A. Biochem. J. 1968; 109: 669-672Crossref PubMed Scopus (214) Google Scholar, 4Yariv J. Kalb A.J. Levitzki A. Biochim. Biophys. Acta. 1968; 165: 303-305Crossref PubMed Scopus (186) Google Scholar, 5Brewer C.F. Brown R.D.I. Koenig S.H. J. Biomol. Struct. Dyn. 1983; 1: 961-997Crossref PubMed Scopus (59) Google Scholar). The three-dimensional structure of the lectin at 1.75-Å resolution has been determined by x-ray diffraction analysis (6Hardman K.D. Agarwal R.C. Freiser M.J. J. Mol. Biol. 1982; 157: 69-86Crossref PubMed Scopus (157) Google Scholar), and a complex with Me-α-Man to 2.9-Å resolution (7Derewenda Z. Yariv J. Helliwell J.R. Kalb A.J. Dodson E.J. Papiz M.Z. Wan T. Campbell J. EMBO J. 1989; 8: 2189-2193Crossref PubMed Scopus (267) Google Scholar). Recently, the x-ray crystal structure of ConA bound with methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside to 2.3 Å has been reported (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Early studies by Goldstein and co-workers (9Goldstein I.J. Poretz R.D. Liener I.E. Sharon N. Goldstein I.J. The Lectins. Academic Press, Inc., New York1986: 35-244Google Scholar) established that ConA has specificity for α-pyranose forms of Glc and Man, which contain similar hydroxyl group configurations at the 3-, 4-, and 6-positions. Monodeoxy derivatives of the monosaccharides at these positions showed essentially complete loss of binding to the lectin, thus establishing the specificity of the so-called "monosaccharide-binding site" in ConA. Later studies demonstrated that certain N-linked oligomannose and complex-type oligosaccharides possessed much higher affinities (∼50-fold or greater) than the monosaccharide methyl-α-D-mannopyranoside (Me-α-Man) (10Bhattacharyya L. Brewer C.F. Eur. J. Biochem. 1989; 178: 721-726Crossref PubMed Scopus (47) Google Scholar), thus suggesting extended site binding interactions with the lectin. The trisaccharide moiety 3,6-di-O-(α-D-mannopyranosyl)-D-mannose, which is part of all N-linked oligosaccharides, was shown to bind with nearly 100-fold higher affinity than Me-α-Man, and to induce conformational changes in ConA similar to those of the larger N-linked carbohydrates (10Bhattacharyya L. Brewer C.F. Eur. J. Biochem. 1989; 178: 721-726Crossref PubMed Scopus (47) Google Scholar, 11Brewer C.F. Bhattacharyya L. J. Biol. Chem. 1986; 261: 7306-7310Abstract Full Text PDF PubMed Google Scholar). These results indicated that the trimannosyl moiety in N-linked carbohydrates was primarily responsible for their high affinity binding to ConA.Detailed insights into the specificity of carbohydrate-protein interactions requires not only relative binding affinity data, but also thermodynamic data to establish whether extended binding site interactions occur. We recently described titration microcalorimetry studies of the binding of methyl-3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside (1) (Fig. 1) to ConA (12Mandal D.K. Kishore N. Brewer C.F. Biochemistry. 1994; 33: 1149-1156Crossref PubMed Scopus (262) Google Scholar). The results showed that 1 possesses nearly 6 kcal mol−1 greater change in binding enthalpy (−ΔH) than Me-α-Man, thus providing direct evidence for extended recognition site by the lectin of the trimannoside epitope. Similar studies with deoxy analogs of the α(1-3)Man residue of the trimannoside established that the 3-OH on this arm is required for high affinity binding (13Mandal D.K. Bhattacharyya L. Koenig S.H. Brown III, R.D. Oscarson S. Brewer C.F. Biochemistry. 1994; 33: 1157-1162Crossref PubMed Scopus (68) Google Scholar).In the present study a series of monodeoxy derivatives of the α(1-6) arm and "core" Man residue of the trimannoside, as well as dideoxy and trideoxy analogs of 1 (Fig. 1), were synthesized and their binding to ConA investigated by isothermal titration microcalorimetry measurements. In light of the recently reported x-ray crystal structure of ConA and the trimannoside (8Naismith J.H. Field R.A. J. Biol. Chem. 1996; 271: 972-976Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), the present results provide information on the energetics of binding of the trimannoside to ConA, and, in turn, on the structure of the complex in solution.
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