Indian Journal of Biochemistry & Biophysics Vol. 37, October 2000, pp. 299-306

Binding mechanism of methyl-a-N--acetyl-D-galactopyranosyl amine to Artocarpus lakoocha lectin, artocarpin: A proton nuclear magnetic

resonance study

S K Rasidul Amin 1, Subhasis Banerjee', Sitapati R Kasturi2 and Bishnu P Chatterjee'*

'*Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jada~pur, Calcutta 700 032 and

1'ata Institute of Fundamental Research, Colaba, Mumbai 400 005

Received 3 February 2000; revised 30 June 2000

The dynamics of the binding mechanism between Artocarpus lakoocha lectin and Me-a-o-GalNAc has been studied using 1H NMR spectroscopy. Various thermodynamic parameters have been calculated with the help of temperature dependence of line broadening of the methoxy group resonance of Me-a-o-GalNAc. No change in the chemical shift has been observed while full line width at half height of the sugar protons was found to increase with increasing temperature indicating that the binding ligand is in fast exchange. No chemical shift between bound and free ligands has been observed. The activation parameters obtained from the association and dissociation rate constants suggest that the association process is controlled by high activation entropy which is due to the specific orientation of both lectin and sugar whereas the contribution of activation enthalpy is small. On the other hand, the dissociation reaction is controlled by high activation enthalpy due to the break in the interaction between the sugar and the lectin. From NMR data a two-step binding mechanism has been proposed. The associated complex is stabilized mainly by hydrogen bonding and van der Waals attractions while hydrophobic interaction is not significant as indicated by the negative entropy and enthalpy values.

Lectins are a class of multivalent proteins or glycoproteins of non-immune origin, which bind specifically and reversibly with simple or complex carbohydrates. Though their physiological roles in vivo are not fully understood, it is anticipated that lectins are involved in cell-cell interactions and thus are widely used as a probe to find out the carbohydrate structures on cell surfaces 1• The physicochemical nature of the binding sites of most of them is not fully characterized. The molecular mechanism of lectin-sugar interactions have been probed by different techniques viz., fluorescence spectroscopy, stop-flow measurement, T-jump relaxation study and UV difference spectroscopy2-6. In these experiments, a fluorophoric or chromophoric group which might influence the binding specificities of lectins is attached to the sugar molecule. So, the presence of these sterically hindered groups and their

*To whom correspondence may be addressed. E-mail: bcbpc@mahendra.iacs.res.in Abbreviations used : AlA, Artocarpus integrifolia agglutinin; ALL, Artocarpus lakoocha lectin ; PNA, Peanut agglutinin; WGA, Wheat germ agglutinin; Me-a-o-GalNAc, Methyl-a-N-acetyl­o-galactopyranosylamine; Me-a-D-Gal , Methyl-a-o-galactopyra­noside; NBS, N-bromosuccinimide.

additional entropic contribution affect the results of lectin-sugar interactions. On the other hand, NMR spectroscopy is a useful tool to calculate various physical parameters from the change in chemical shift/or linewidth of different sugar protons during their binding equilibrium with lectins7

.16

. Also binding mechanism and topology of lectin-sugar ' interactions can be understood from a study of NMR spectroscopy.

Artocarpin, a purified Artocarpus lakoocha lectin (ALL) is a glycoprotein containing II% of sugar17

and the binding of ALL was found to be specific for Me-a-D-Gal/Me-a-D-GalNAc and oligosaccharides conta1mng a-1 ,6-linked nonreducing galactosyl residue 18

'19

• In the present paper we have discussed the possible mechanism of binding between ALL and Me-a-D-GalNAc which is based on thermodynamic and kinetic parameters calculated from the change in the line width of the resonance of methoxy protons on binding to ALL using 1H NMR spectroscopy.

Materials and Methods Me-a-D-Gal, Me-a-D-GalNAc, N-bromosuccini­

mide, melibiose-agarose and D20 (99.96%) were purchased from Sigma Chemical Co., St. Louis, USA.

300 INDIAN J BIOCHEM BIOPHYS, VOL. 37, OCTOBER 2000

All other reagents were of highest analytical grade and obtained from local agencies.

Purification of artocarpin The .lectin from Artocarpus lakoocha seed was

purified -by affinity chromatography on melibiose­agarose column as described previously 17

.

Chemiuil modification of artocarpin Modification of tryptophan residues in artocarpin

was performed by oxidation with N-bromo­succinimide as described earlier20

.

Preparation of NMR sample Deulerated phosphate buffered saline (PBS) was

prepared by repeated exchange with 0 20. Different concen[rations of artocarpin and Me-a-o-GalNAc were prepared in 10 mM deuterated PBS containing 0.02% sodium azide. Lyophilized artocarpin was equilibrated for several hours at 25°C in 0 20 to reduce the size of the residual HDO peak in subsequent NMR studies and lyophilized. The process was repeated three times. Three solutions of artocarpin with different protein concentrations, viz ., 0.045, 0.057 and 0.065 mM were prepared.

The ligand was exchanged three times with 0 20. Lectin sample in 0 20-PBS was mixed with different concentrations of ligand by gradual addition of the stock solution .

NMR measurements 1H I\'MR spectroscopy was performed on a Bruker

AMX-500 MHz FT-NMR spectrometer which was operated using 5 kHz spectral width, 5 sec relaxation delay, 8 K data points and the number of scans depending on sugar concentrations . For a particular sugar concentration, NMR instrument was operated at different temperatures, viz., 10°, 15°, 20°, 25° and 30°C. All experiments were performed with suppression of the HDO resonance by homogated decoupiing. The linewidths (~v 1 n) at half-height were measured by Lorentzian fit and the measured linewidths of different sugar protons were corrected for magnetic field inhomogeneity by using the linewidth at half-height of internal acetone resonance as the correction factor. Sample volumes ranging from 525-800 1-11 were analyzed in 5 mm NMR tubes .

Control experiments Three control experiments were performed.

Linewidth of Me-a-o-GalNAc protons were

measured (i) in the presence of excess competing sugar, Me-a-o-Gal, (ii) with two different protein concentrations (0.045 and 0.065 mM) of ALL and (iii) in the presence of 4 mM EDTA under same experimental conditions.

Analysis of NMR results 1H spectra of the -OCH3 protons of methyl-a-o­

GalNAc in the presence and absence of ALL are presented in Fig. lb and I a respectively, which shows that the linewidth of -OCH3 protons increased due to binding of the sugar to ALL and there was no proton chemical shift. When a sugar molecule is in binding equiiibrium with the lectin, the NMR peaks of the

I 3.5

(b)

(a)

ppm

I 3.0

Fig. l-1H NMR spectrum of -OCH3 group of Me-a-o-GalNAc (3.4 mM) (a), in the free state; (b) , in the presence of ALL (0.057 mM) at I ooc

AMIN et_ql.: BINDING MECHANISM OF METHYL-a-N-ACETYL-o-GALACTOPYRANOSYL AMINE TO LECTIN 30 I

sugar protons may shift and/or broaden due to chemical exchange of sugar molecule between bound and free environments . The observed change in the resonance frequency (~CO..) and spin-spin relaxation rate (l/T2p) due to binding of a small molecule to a macromolecule can be described by the Swift and Connick relations21

. Since in the present case no chemical shift has been observed between the bound and free ligand, the spin-spin relaxation rate (l/T2p) can be described by:

f ... (I)

In eqn. (I) I/T2p is the spin-spin relaxation rate of the ligand protons observed due to binding of the

0.7...------------,-----,

0.6

f '-' 0.5

..!?< <l

0.4

Fig. 2-Change of the line broadening of the methoxy resonance of Me-a-o-GaiNAc (0.97 mM) as a function of temperature in the presence of ALL (0.057 mM)

ligand to the protein, f is the fraction of the ligand bound to the protein, 'tM is the residence time of the ligand on the protein and T2M is the spm-spm relaxation time in the bound environment.

Results and Discussion In the present study, it was observed that ~v

increased (Fig. 2) with increase in temperature indicating a slow exchange of the sugar between bound and free environments. This suggests that the change in relaxation rate is governed only ~y the exchange reaction rate which is slow ('tM >>T2M) and hence T2M can be neglected in equation I.

. .. (2)

Therefore, different equilibrium and kinetic parameters could be determined from the temperature dependence of the line-broadening of particular sugar protons in the presence of lectin.

Several control experiments were carried out in order to ensure that the line broadening of sugar protons was due to specific binding to lectin. It was observed that (i) the reciprocal of the linewidth, 1/~v was a linear function of sugar concentration, [Sh (Fig. 3); (ii), in the presence of excess competing sugar, Me-a-D-Gal, the line broadening was diminished; (iii), line broadening was unaffected with change in ALL concentrations; (iv), in the presence of 4 mM EDTA linewidth remained unchanged and (v), in the presence of chemically modified ALL no change in line width was observed (not shown). From the foregoing control experiments, it could be concluded that the line broadening was not due to change in viscosity developed upon addition of ALL or due to the presence of metal ions in ALL 1

R. Hence it is clear that the observed line broadening is due to specific binding of Me-a-o-GalNAc to the lectin , artocarpin.

Since in our present study, Me-a-o-GaiNAc is in equilibrium with ALL, it is possible to calculate various physical parameters . In the equilibrium state between the sugar, Sand the lectin , P

k,

S+P SP

As the sugar was present in excess compared to the lectin , [S]>>[P] then it can be shown using Michaelis-Menten equation that

302 INDIAN J BIOCHEM BIOPHYS, VOL. 37, OCTOBER 2000

8

4 -1

1/t:..-\)(Hz )

8

-6~----------------------------~

Fig. 3-Piot of the total sugar concentration , [Sh vs the reciprocal of the ~ange of linewidth , l/6v of methoxy resonance of Me-a­o-GaiNAc for the binding to ALL at different temperatures accordiag to the eqn. (8), [(o), 10°C; (6), I5°C; (0 ), 20°C; (.A.), 25°C; aild (•) 30°C.

[S] j n[P]- K T j D

... (3)

where: [Sh is the total sugar concentration, [P] is the lectin concentration, n is the number of binding sites of ALL tetramer which was found to be two 18

, f is the fraction of sugar bound to ALL and K0 is the equilibrium dissociation constant. Substituting the value off in (3)

[Sh= n [P]T2P- Ko 't"M

. . . (4)

Again ~v, the change in the line width of sugar protons due to interaction with the lectin is given by

... (5)

where ~VF is the linewidth of free sugar and ~v, is the line width of the sugar after interaction with the

Table )-Equilibrium dissociation constant (K0 ), dissociation rate constant (k.J), equilibrium association constant (KA) and association rate constant (k.) for the binding of Me-a-o­GaiNAc to ALL at different temperatures

T K0 x 103 kd KA x 10·2

(oC) (M) (s·l) (M ·I)

10 2.50 3 1.90 3.99

15 2.71 39.56 3.69

20 3.06 59.28 3.27

25 3.99 83.96 2.50

30 4.94 107.30 2.02

k. xl0·4

(M·Is· l)

1.27

1.46

1.94

2.1 0

2.17

Estimated errors: K0 , KA (7.7 -15%); kd , (0.3-6.5%) and k. (8-1 6%)

lectin. ~v is related to I /T2r, change in relaxation rate by the equation

. . . (6)

where l/rtT2 is the observed relaxation rate of sugar protons in the presence of lectin and l/rtT2<oJ is the relaxation rate of the free sugar. Eqn. 6 is reduced to

I T2r=-­

Jr~V

Substituting the value of T2r in equation 4

[S] _ _ l_n[P]_K T- D

6v 1rrM

. . . (7)

. .. (8)

Fig. 3 shows the plot of [Sh vs 1/~v · at five different temperatures. 1M, residence time of the sugar on the protein, obtained from the slope is the reciprocal of the dissociation rate constant, llkct. Ko, the equilibrium dissociation constant evaluated from the y-intercept is equal to the reciprocal of the equilibrium association constant, 1/KA. (Thus, kct and KA are evaluated from 1M and K0 respectively). Table I summari zes the values of 1M, Ko, KA and kct at different temperatures. The association equi librium constant, KA is again equal to the ratio of association and dissociation rate constants, KA'=kafkct. The value of k. was calculated for various temperatures and is presented in Table I .

The changes in Gibbs free energy of binding ~G0 ,

enthalpy of binding ~H0 and entropy of binding M 0

are related to K0 by the equation

AMIN et al.: BINDING MECHANISM OF METHYL-a-N-ACETYL-o-GALACfOPYRANOSYL AMINE TO LECTIN 303

-5.2r---r--------------..,

0 :11: -5.6 ..5

0

0

0

3.4 3.5 3.6

lx 103

T Fig. 4-van't Hoff plot of In K0 vs liT for the binding of Me-a-o­

GalNAc to ALL according to the equation (9)

/1Go tJ!o !:!.So InK =--=----

o RT RT R ... (9)

where R is the gas constant and T is the temperature. Fig. 4 shows the vant' s Hoff plot of In K0 vs liT and the I:!.H0 and I:!.S0 obtained from the slope and the intercept are shown in Table 2 which also presents I:!.G0 value. The activation energy for association (E:' A) and dissociation (gJ A) reactions are related to associatiOn and dissociation rate constants respectively by the following equations.

Ea Ink =In A-~

a RT

Ect and In k =In A--A

d RT

... (10)

... (II)

where A is the frequency factor. From the slope -EAIR of the plot, In ka or In kct vs liT (Fig. 5 and 6) the values of EAa and EAd are obtained and presented in Table 2. Finally, the activation parameters !:!.d, Mt and fl.S for the association and dissociation reactions were determined from the plot of In (kal7) vs liT and In (kctl7) vs liT (Fig. 7 and 8) using the equation of

. . h 22 trans1tton state t eory .

. -I:!.H# M# k In (kjT)= +-+-

RT R h ... (12)

where k is the Boltzmann constant and h is the Planck constant; similar equation can be used for kct. The data (t:Jf, !:!.~,!:!.d) obtained are summarized in Tabie 2.

Table 2-Thermodynamic and kinetic parameters for the binding of Me-a-o-GalNAc to ALL

Parameter Value (kcal/mol) L1Gol -3 .27 !J.HO -5.94 /::;.So2 -8.96

TIJ.Sol -2.67

E'A 4.91 ~A 10.85

t!..G.- 11.57 t!..G-d 14.83 t:,}{ 8 a 4.32 t:,}{ # d 10.27 ~2. -24.34 T~.~ -7.25 ~} -15.37

TL\Sd -4.58

1at 25°C; 2 in cal mol' 1

Estimated errors are± I kcal mol' 1, except for!!,.~ A.

t:,H 8d and Tt!..S Nd (± 0.5 kcal mo1' 1

) .

10.0 0

0

9.8

0 .::<

5

9.6

0

9.1.

3.6

Fig. 5-Arhenius plot of In k. vs liT for the binding of Me-a-o­GalNAc to ALL.

304 INDIAN J BIOCHEM BIOPHYS, VOL. 37, OCTOBER 2000

4.6

~" 4.0 c

14

0

0

0

36

Fig. 6-Arhenius plot of In k0 vs liT for the binding of the Me-a­D-GalNAc to ALL.

0

0

3.8

3.6

Fig. 7-Plot of In (k/7) vs liT for the binding of Me-a-o-GalNAc to ALL.

'-1.0

~ -1.6 ...:i'lt--c

-2.2

0

I 1 - x !O' T

3.5

Fig. 8-Plot of In (k0/T) vs liT for the binding of Me-a-D­GaiNAc to ALL.

3.6

It is found from Table l that KA decreases and K0

increases with increase of temperature showing that the affinity of the lectin for the ligand decreases with rise of temperature. This indicates that there is an overall energy barrier characterized by a positive /).Ga# which is due to high activation entropy (t:.Sa#)

and low activation enthalpy (/).H.#). Hence the high activation energy along with the temperature dependence of equilibrium constants suggests that the binding of Me-a-o-GaiNAc to ALL involves a more complex mechanism and cannot be explained by a simple bimolecular mechanism. We propose a two­step process for the binding mechanism of ALL and Me-a-D-Ga ii NAc. An activated complex is formed between Me-a-D-GaiNAc and ALL in the transition state which is then converted to the final complex by intramolecular rearrangement. So, the process can be represented as

k, k2 P+S~Psn~ps ... (13)

This mechanism is valid only if the concentration of the transition state complex PS is very small compared to the final complex, PS and the starting molecules P and S. This is confirmed from the high

AMIN et al.: BINDING MECHANISM OF METHYL-u-N-ACETYL-o-GALACTOPYRANOSYL AMINE TO LECTIN 305

activation energy of dissociation (EA d), which indicates that the Boltzmann distribution between the transition state complex and the final complex at 25°C is

~=e-E~IRT =l.lx1o-s no

where n is the number of molecules in the activated state and n0 is the number of molecules in the associated state_ This indicates the presence of a short-lived transition state complex, Pstt. From eqn. (13)

If the formation of the transition state complex Pstt from the final complex PS is the rate-determination step of dissociation reaction with the assumption that k2>>k_2 then the residence time of the sugar on the

lectin can be written as 'tM= 1/kd= l/k_2. The apparent association constant according to eqn . ( 13) is

(since K 1 is small)

and the apparent association rate constant (k,) becomes k,=K1 K2k_2=K1k2_ It can be seen from Table 1 that the association constant decreases with rise of temperature whereas the association rate constant increases with increase in temperature. In order to explain the temperature dependence of association rate constant (here K1k2) derived from eqn. (13) relating to the binding mechanism, it can be concluded that k2 increases more rapidly with increase in temperature whereas K1 decreases less rapidly with increasing temperature. The net result is that the association rate constant increases with increasing temperature. This also indicates that the activation enthalpy for the dissociation reaction , !:l.H/ is greater than that of the enthalpy change /:l.H0

•

From the foregoing results, it can be concluded that the binding between ALL and Me-a-o-GalNAc takes place in two steps. The first step is probably a diffusion controlled reaction which occurs due to a loose association of, the sugar to the binding site of

the lectin (P .. . S). This step is associated with a low enthalpy change. The second step is a mutual fitting of the sugar to the binding site of the lectin by intermolecular rearrangement. The second step is associated with a high negative entropy of activation due to requirement of a particular conformation of the lectin binding site and the sugar molecule. On the other hand, dissociation of the final complex is associated with a large activation enthalpy (H/) which is prerequisite for breaking the interaction between the sugar and the lectin binding site. The total binding process is represented schematically in Fig. 9 showing thermodynamic and kinetic para­

meters at 25°C. By analysis of several protein-protein and protein­

ligand binding reactions, Ross and Subramanian23

concluded that almost all association processes could occur due to hydrogen bonding and van der Waals attractions. This has been observed when the values for both enthalpy change (!:l.H0

) and entropy change (/:l.S0

) are negative in sign. On the other hand, hydrophobic and ionic interactions are characterized

by positive !:l.H0 and !:l.S0 values. Our results on the interaction between ALL and Me-a-o-GalNAc are similar to other lectin-sugar interactions (Table 3) suggesting that this is due to intermolecular hydrogen bonding and van der Waals attractions. At first a partially immobilized complex is formed in the

15 p:;-:f

n"~- i * - r c.sd 12

1 'i"= 9 """ __ 1 ---------lol E 0 u

1 -"' :6 t.H: ~

1 c,Hj "' c

w

j P+S

0 PS

React ion Coordinate

Fig. 9-Schematic diagram for the change in thermodynamic and kinetic parameters for the binding of Me-u-o-GalNAc to ALL at 25°C

306 INDIAN J BIOCHEM BIOPHYS, VOL. 37, OCTOBER 2000

Table 3-Comparison of thermodynamic and kinetic parameters for different lectin-sugar interaction

Parameter Value (kcal mol- 1)

ALL-Mea-o-GalNAc PNA- Mea-o-Gal" WGA-3 NeuLach AlA- Mea-o-GalNAc"

6G0 -3.27 -4.4 -3 .8 -3.63

Mr -5 .94 -9 .9 -13.3 -8 .91

·TM0 2.67 5.6 9.5 5.07

6d. 11 .57 11.4 9.6 10.43

6du 14.83 15.5 13.4 14.06

Mfl. 4.32 0 4 .1 2.07

Mflu 10.27 8.9 17.4 10.95

-nstt. 7.25 11.4 5.5 8.36

T6sHu 4.58 6.6 -4.0 3.28

"Peanut agglutinin-Me-a-o-galactopyranoside7; hWheat germ agglutinin-a, 2-3 isomer of N-acetylneuraminyllactose 12

; "Artocarpus in.tegrifolia aggl utinin-aN-acetyl D-galactosamine 14

activated state characterized by a negative !:J.Sa#. whereas if the association process would have occurred via hydrophobic interactions, then !:J.Sa# would have shown a posi tive value23

. Also, Table 3 indicates that most of the lectin-sugar interactions are controlled by the activation entropy for the association step and activation enthalpy for the dissociation reaction . In summary, the important stabilizing factors of lectin-sugar complexes seem to be hydrogen bonding and van der Waals interaction while hydrophobic effect is less significant.

Acknowledgement One of us (SRA) was supported by a fellowship

from Council of Scientific and Industrial Research, New Delhi. We thank Prof. Anil Saran and Mrs . Mamata Prachand of Tata Institute of Fundamental Research, Mumbai for ass istance .

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