Thermodynamics of Lectin-Carbohydrate Interactions
BINDING OF THE CORE TRIMANNOSIDE OF ASPARAGINE-LINKED CARBOHYDRATES AND DEOXY ANALOGS TO CONCANAVALIN A*

(Received for publication, September 11, 1996, and in revised form, December 18, 1996)

Dipti Gupta Dagger , Tarun K. Dam Dagger , Stefan Oscarson § and C. Fred Brewer Dagger

From the Dagger  Departments of Molecular Pharmacology, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 and the § Department of Organic Chemistry, Stockholm University, Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The trisaccharide 3,6-di-O-(alpha -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-alpha -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 alpha (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 alpha (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 alpha (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 alpha (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).


INTRODUCTION

The ability of concanavalin A (ConA)1 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 (1, 2). 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) (3-5). The three-dimensional structure of the lectin at 1.75-Å resolution has been determined by x-ray diffraction analysis (6), and a complex with Me-alpha -Man to 2.9-Å resolution (7). Recently, the x-ray crystal structure of ConA bound with methyl-3,6-di-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside to 2.3 Å has been reported (8).

Early studies by Goldstein and co-workers (9) established that ConA has specificity for alpha -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-alpha -D-mannopyranoside (Me-alpha -Man) (10), thus suggesting extended site binding interactions with the lectin. The trisaccharide moiety 3,6-di-O-(alpha -D-mannopyranosyl)-D-mannose, which is part of all N-linked oligosaccharides, was shown to bind with nearly 100-fold higher affinity than Me-alpha -Man, and to induce conformational changes in ConA similar to those of the larger N-linked carbohydrates (10, 11). 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-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside (1) (Fig. 1) to ConA (12). The results showed that 1 possesses nearly 6 kcal mol-1 greater change in binding enthalpy (-Delta H) than Me-alpha -Man, thus providing direct evidence for extended recognition site by the lectin of the trimannoside epitope. Similar studies with deoxy analogs of the alpha (1-3)Man residue of the trimannoside established that the 3-OH on this arm is required for high affinity binding (13).


Fig. 1. Structures of oligosaccharides 1-14. Man, GlcNAc, Gal, Glc, 2-dMan, 3-dMan, 4-dMan, and 6-dMan represents mannose, N-acetylglucosamine, galactose, glucose, 2-deoxymannose, 3-deoxymannose, 4-deoxymannose, and 6-deoxymannose residues, respectively.
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In the present study a series of monodeoxy derivatives of the alpha (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 (8), 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.


EXPERIMENTAL PROCEDURES

Materials

ConA was prepared from Jack bean (Canavalia ensiformis) seeds (Sigma) according to the method of Agrawal and Goldstein (14). The concentration of ConA was determined spectrophotometrically at 280 nm using Acm1% = 13.7 at pH 7.2 (9) and expressed in terms of monomer (Mr = 25, 600). Me-alpha -Man and 1 were purchased from the Sigma. Synthesis of trimannoside derivatives 2-13 in Fig. 1 has been reported (15). The synthesis of 14 will be described elsewhere. The concentrations of carbohydrates were determined by modification of the Dubois phenol-sulfuric acid method (16, 17) using appropriate monosaccharides (Man, 2-deoxymannose, 3-deoxymannose, 4-deoxymannose, and 6-deoxymannose) as standards.

Titration Microcalorimetry

Isothermal titration microcalorimetry was performed using an OMEGA Microcalorimeter from Microcal, Inc. (Northampton, MA). In individual titrations, injections of 4 µl of carbohydrate were added from the computer-controlled 250-µl microsyringe at an interval of 4 min into the lectin solution (cell volume = 1.3424 ml) dissolved in the same buffer as the saccharide, while stirring at 350 rpm. An example of an isothermal titration microcalorimetry experiment is shown in Fig. 2 for trisaccharide 13 with ConA at 27 °C. Control experiments performed by making identical injections of saccharide into a cell containing buffer with no protein showed insignificant heats of dilution. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal, with Delta H (enthalpy change in kcal mol-1), Ka (association constant in M-1) and n (number of binding sites per monomer), as adjustable parameters. The quantity, c = Ka Mt(0), where Mt(0) is the initial macromolecule concentration, is of importance in titration microcalorimetry (18). All experiments were performed with c values 1 < c < 200. The instrument was calibrated using the calibration kit containing ribonuclease A (RNase A) and cytidine 2'-monophosphate (2'-CMP) supplied by the manufacturer. Thermodynamic parameters were calculated from,
&Dgr;G=&Dgr;H−T&Dgr;S=−RT<UP> ln</UP>K<SUB>a</SUB> (Eq. 1)
where Delta G, Delta H, and Delta S are the changes in free energy, enthalpy, and entropy of binding. T is the absolute temperature and r = 1.98 cal mol-1 K-1.


Fig. 2. Calorimetric titration of ConA with trisaccharide 13 at 27 °C. Top, data obtained for 50 automatic injections, each 4 µl, of 13; and bottom, the integrated curve showing experimental points (black-square) and the best fit (-). The buffer was 0.1 M HEPES containing 0.9 M NaCl, 1 mM MnCl2, and 1 mM CaCl2 at pH 7.2. For details see "Experimental Procedures."
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RESULTS AND DISCUSSION

Our 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 (-Delta H) and 60-fold higher affinity than Me-alpha -Man (Table I) (12). 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 (8).

Table I.

Thermodynamic parameters derived from the titration of ConA at pH 7.2 with saccharides at 27 °C

The buffer was 0.1 M HEPES containing 0.9 M NaCl, 1 mM Mn2+, and 1 mM Ca2+ at pH 7.2. Values of n were between 0.95 and 1.09 in all cases.
Carbohydrate (abbr.) Carbohydrate Lectin Kaa  -Delta G  Delta Delta G  -Delta Hb  Delta Delta (H)c  -TDelta Sb

mM M-1 × 10-4 kcal mol-1
Mealpha Man 46.0 0.48 0.82d 5.3 2.5 8.2 6.2 2.9
 1 "Trimannoside" 7.0 0.13 49.0d 7.8 14.4 6.6
 2 alpha (1-3)2-deoxy 6.2 0.14 56.8d 7.9  -0.1 14.5  -0.1 6.6
 3 alpha (1-3)3-deoxy 9.0 0.14 5.39d 6.5 1.4 11.0 3.4 4.5
 4 alpha (1-3)4-deoxy 9.0 0.24 9.2 6.8 1.0 12.3 2.1 5.5
 5 alpha (1-3)6-deoxy 6.4 0.15 39.6d 7.7  -0.1 14.0 0.4 6.3
 6 alpha (1-6)2-deoxy 6.2 0.23 50.1 7.8 0.0 14.9  -0.5 7.1
 7 alpha (1-6)3-deoxy 14.5 0.33 3.88 6.3 1.5 11.2 3.2 4.9
 8 alpha (1-6)4-deoxy 7.0 0.23 2.54 6.0 1.8 11.7 2.7 5.7
 9 alpha (1-6)6-deoxy 12.5 0.33 3.01 6.1 1.7 11.6 2.8 5.5
10 "core"2-deoxy 12.5 0.24 11.7 6.9 0.9 13.4 1.0 6.5
11 "core"4-deoxy 9.3 0.26 4.63 6.4 1.4 12.1 2.3 5.7
12 alpha (1-3)3-deoxy, core 2-deoxy 42.5 0.54 3.60 6.2 1.6 10.6 3.8 4.4
13 alpha (1-3)3-deoxy, core 4-deoxy 7.0 0.24 1.93 5.9 1.9 9.7 4.7 3.8
14 alpha (1-3)3-deoxy, core 2,4-deoxy 45.0 0.75 0.99 5.5 2.3 8.7 5.7 3.2

a Errors in Ka values were between 2 and 10%.
b Errors in Delta H and TDelta S were ±0.1 to ±0.2.
c Relative to 1.
d Data taken from Mandal et al. (13) and included here for comparison.

Binding of Monodeoxy Analogs of Trimannoside 1

Our previous studies examined the thermodynamics of binding of a series of monodeoxy analogs of 1 possessing substitutions on the alpha (1-3) arm (13). 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, -Delta H and entropy (TDelta S) 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 -Delta H 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 -Delta H with respect to 1 (Table I). Thus, the 3- and 4-hydroxyl groups on the alpha (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 alpha (1-6) arm possessed decreased affinities and -Delta H values relative to 1 (13). These results provided evidence that the alpha (1-6)Man residue binds to the so-called "monosaccharide"-binding site of ConA, by analogy to the requirements of monosaccharide binding to ConA (9). However, in order to directly determine the interactions of the alpha (1-6) arm of 1 with ConA, the 2- (6), 3- (7), 4- (8), and 6-deoxy (9) derivatives of the alpha (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 alpha (1-6) arm. On the other hand, the Ka and Delta H values for 7, 8, and 9 are significantly lower than that of 1, indicating the involvement of 3-, 4-, and 6-OH of the alpha (1-6)Man residue of the trimannoside in binding (19). The magnitude of the reductions in Ka and Delta H 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 (9), the data are consistent with binding of the alpha (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 -Delta H 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 1

The 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 Delta H 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 Delta H 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 Delta H 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 Delta H 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 alpha (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 Delta H of 14 is -8.7 kcal mol-1 which is 5.7 kcal mol-1 less than that of 1. This reflects losses in Delta H of the corresponding monodeoxy analogs 3, 10, and 11, relative to 1.

Comparison with the X-ray Crystal Structure of 1 Complexed to ConA

The x-ray structure of the complex formed by the free sugar of 1 with ConA has recently been reported (8). 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 alpha (1-6)Man residue of 1 binds via its 3-, 4-, and 6-hydroxyl groups in the same manner as Me-alpha -Man in its crystalline complex with the lectin (7). 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 alpha (1-3) Man residue to the N-H and side chain O of Thr-15, and the 4-OH of the alpha (1-3)Man residue to the side chain -OH of Thr-15 (8) (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 (8). 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 (8).


Fig. 3. View of the free sugar of trimannoside 1 (no alpha -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 alpha (1-6)Man by 6 and the alpha (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 (8).
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Table II.

Hydrogen bonding and polar contact (<3.5 Å) distance between the free sugar of trimannoside 1 and ConA as determined by x-ray crystallographya

Data are taken from Ref. 8.
Sugar Protein Distance

Å
 alpha (1-6)Man
  O-3 Arg-228 N 2.9
  O-4 Asn-14ND2 2.9
  O-4 Asp-208OD1 2.7
  O-4b Arg-228 N 3.5
  O-5 Leu-99 N 2.9
  O-6 Asp-208OD2 2.9
  O-6 Tyr-100 N 3.1
  O-6b Leu-99 3.1
Central Man
  O-2 OWb 2.6
  O-2b Asp-16OD1 3.1
  O-4 Tyr-12 OH 2.8
 alpha (1-3)Man
  O-3 Thr-15 N 2.8
  O-3 Thr-15OG1 2.9
  O-3b Pro-13 O 2.9
  O-4 Thr-15OG1 3.1
  O-4b Asp-16 N 3.0

a 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.

Nonlinearity of the Delta Delta H and Delta Delta G Values of the Individual Hydroxyl Groups of 1

The present thermodynamic data indicate that the Delta Delta H values for the monodeoxy analogs in Table I are nonlinear. For example, the sum of the Delta Delta H values for monodeoxy analogs 7, 8, and 9 is ~-10.5 kcal mol-1 (Table I). To a first approximation, this represents the combined Delta H contribution of the 3-, 4-, and 6-OH of the alpha (1-6)Man residue of 1 at the monosaccharide site of the lectin. This value can be compared to the Delta H for Me-alpha -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 Delta H contribution of the ring oxygen of the alpha (1-6)Man residue of 1 (Fig. 3) which would make this value even greater and hence the difference with the Delta H of Me-alpha -Man larger. Similarly, the combined Delta Delta H values for the 3-OH and 4-OH of the alpha (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 Delta H between 1 and Me-alpha -Man of -6.2 kcal mol-1 which reflects binding of the alpha (1-3)Man and the central Man residues of 1. Furthermore, the sum of the Delta Delta H values for the 3-, 4- and 6-OH of the alpha (1-6)Man residue (7, 8, and 9), the 3- and 4-OH of the alpha (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 Delta H for 1 of -14.4 kcal mol-1. Thus, the sum of the Delta Delta H values for the hydroxyl groups of 1 obtained from the monodeoxy analogs in Table I does not correspond to the measured Delta H of 1. In all of the above cases, the sum of the Delta Delta H values for specific hydroxyl groups on certain Man residues of 1 obtained from the corresponding monodeoxy analogs is greater than the measured Delta H for that residue(s).

This nonlinear relationship in Delta Delta H is also present in the di- and trideoxy analogs of 1. Delta Delta H for dideoxy analog 12 is -3.8 kcal mol-1 as compared to the sum of the Delta Delta H values for corresponding monodeoxy analogs 3 and 10 of -4.4 kcal mol-1. Likewise, Delta Delta H for dideoxy analog 13 is -4.7 kcal mol-1 as compared to the sum of the Delta Delta H values for corresponding monodeoxy analogs 3 and 11 of -5.7 kcal mol-1. In the case of trideoxy analog 14, its Delta Delta H value is -5.7 kcal mol-1 as compared to -6.7 kcal mol-1 for 3, 10, and 11. Thus, the Delta Delta H values for the monodeoxy analogs are nonlinear in terms of their contributions to the Delta Delta H values for the two dideoxy analogs and the trideoxy analog.

The same nonlinearity is also present in the Delta Delta G values of the monodeoxy analogs. For example, the sum of the Delta Delta G values for 3, 4, 10, and 11 is -4.7 kcal mol-1, however, the difference in Delta Delta G between 1 and Me-alpha -Man is -2.5 kcal mol-1 (Table I). In addition, the sum of the Delta Delta G values for 3 and 10 is -2.3 kcal mol-1 while the Delta Delta G for 12, the corresponding dideoxy analog, is -1.6 kcal mol-1. The sum of the Delta Delta G values for 3 and 11 is -2.8 kcal mol-1 while the Delta Delta G for 13, the corresponding dideoxy analog, is -1.9 kcal mol-1. The sum of the Delta Delta G values for 3, 10, and 11 is -3.7 kcal mol-1 while the Delta Delta G value for 14, the corresponding trideoxy analog, is -2.3 kcal mol-1. Thus, the Delta Delta G values for the monodeoxy analogs are nonlinear in terms of their contributions to the Delta Delta G values for the two dideoxy analogs and the trideoxy analog.

The Delta Delta H and Delta Delta G 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, Delta Delta H 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 alpha (1-6)Man residue and at least two strong H-bonds each to the 4-OH and 6-OH of the alpha (1-6)Man residue. Thus, the Delta Delta H of -3.2 kcal mol-1 for 7 reflects the loss of a single H-bond with the protein, however, the Delta Delta H 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 Delta Delta H 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 alpha (1-3)Man residue. Thus, the Delta Delta H 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 Delta Delta G 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 (20). The data in Table I, however, indicate no such relationship in the free energy difference (Delta Delta G) of monodeoxy analogs that represent the loss of one or more H-bonds such as versus 8 and 9.

The presence of nonlinear relationships in the Delta Delta H and Delta Delta G 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 Delta Delta H and Delta Delta G 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 Delta H of binding of 1 to ConA (21). 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 Compensation

Enthalpy-entropy compensation plots have previously been observed for carbohydrate interactions with lectins (22, 23) and antibodies (24-26), and attributed to the unique properties of water (23). A plot of the -Delta H versus -TDelta S 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 (27, 28), in contrast to antibody-carbohydrate interactions where the slope is often less than unity (25, 26). 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. 4. Plot of -Delta H versus -TDelta S for the binding of ConA to 3,6-di-O-(alpha -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. 12 and 13 and the present study.
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Summary

The 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 alpha (1-6)Man, the 2- and 4-OH groups of the core Man, and the 3- and 4-OH on the alpha (1-3) an of 1 to the lectin. These results agree with the recently described x-ray crystal structure of the complex (8).

Nonlinear effects in the Delta Delta H and Delta Delta G 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.


FOOTNOTES

*   This work was supported by Grant CA-16054 and Core Grant P30 CA-13330 from the National Cancer Institute, Department of Health, Education and Welfare. The NMR facility at AECOM was supported by Instrumentation Grant I-S10-RR02309 from the National Institute of Health and DMB-8413723 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 718-430-2227; Fax: 718-430-8922.
1   The abbreviations used are: ConA, concanavalin A, lectin from jack bean; N-linked, asparagine-linked; Me-alpha -Man, methyl-alpha -D-mannopyranoside; 1, methyl-3,6-di-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 2, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(2-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 3, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(3-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 4, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(4-deoxy-alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 5, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(6-deoxy-alpha -Dmannopyranosyl)-alpha -D-mannopyranoside; 6, methyl-6-O-(2-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 7, methyl-6-O-(3-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 8, methyl-6-O-(4-deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 9, methyl-6-O-(6deoxy-alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-alpha -D-mannopyranoside; 10, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-2-deoxy-alpha -D-mannopyranoside; 11, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(alpha -D-mannopyranosyl)-4-deoxy-alpha -D-mannopyranoside; 12, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(3-deoxy-alpha -D-mannopyranosyl)-2-deoxy-alpha -D-mannopyranoside; 13, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(3-deoxy-alpha -D-mannopyranosyl)-4-deoxy-alpha -D-mannopyranoside; 14, methyl-6-O-(alpha -D-mannopyranosyl)-3-O-(3-deoxy-alpha -D-mannopyranosyl)2,4-dideoxy-alpha -D-mannopyranoside.

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