E L S E V I E R Biochimica et Biophysica Acta 1206 (1994) 215-224

BB Biochi ~mic~a et Biophysica AEta

Modes of association of concanavalin A with a-D-glycosides

Anas t a s s io s T ro g an i s , Char ik l i a I. S t a s s i n o p o u l o u *

Institute of Biology, NCSR 'Demokritos ', 153 10 Aghia Paraskevi, Greece

Received 28 October 1993; revised 14 February 1994

Abstract

Complexes of Con A with a-D-glycosides were studied using 1H-NMR, ESR and fluorescence methods. Correlation times, ~'c, for the interaction of the aglycon protons with the manganese ion, present at the S1 site of the protein, were calculated from T1 measurements at two frequencies. The protons of aromatic aglycons have ~'c values comparable to the rotational correlation time of the protein molecule, whereas those of non-aromatic aglycons have zcs 10 to 100 times lower. The correlation times were combined with the experimentally acquired paramagnetic contributions to proton relaxation due to the presence of the manganese ion to yield manganese-proton distances. These distances show that aromatic aglycons have additional favorable contacts with the protein which stabilize the lectin-saccharide interaction. The results are compared to the crystal structure of the methyl a-D-glycopyranoside complex with Con A and to models earlier proposed for the binding of monosaccharides to Con A.

Key words: Concanavalin A; a-D-Glycoside; Monosaccharide binding; NMR, -1H; Correlation time

1. Introduction

Concanavalin A (Con A) is a legume lectin with well known and very important biological properties which make it an invaluable tool in biochemical research [1]. These properties are derived from its ability to form complexes with derivatives of the specific sugars a-D- mannose and a-o-glucose. The sugars bind to Con A mostly through their terminal units [2]. In glycosides the binding constant for the Con A-sugar complex depends on the nature of the aglycon [3].

Association constants of carbohydrates to Con A have been obtained from UV spectroscopy by a technique based on their ability to displace 4-nitrophenyl a-D-mannopyra- noside from its complex with the lectin [4] and from

* Corresponding author. E-mail: stassin@cyclades.nrcps.ariadne-t.gr and cstas@isosun.ariadne-t.gr. Fax: +30 165 11767. Abbreviations: Con A, concanavalin A; a-MM, methyl a-D-mannopyra- noside; a-MG, methyl a-D-glucopyranoside; a-MUM, 4'-methylumbei- liferyl a-O-mannopyranoside; a-MUG, 4'-methylumbelliferyl a-D-gluco- pyranoside; a-PNM, 4'-nitrophenyl a-D-mannopyranoside; a-PNG, 4'- nitrophenyl a-a-glucopyranoside; a-PG, 2',2',6',6'-tetramethylpiperidin- 4'-yl a-D-glucopyranoside; a-TG, 2',2',6',6'-tetramethylpiperidin-4'-yl- l'-oxyl ot-o-glucopyranoside; TEMPO, 2,2,6,6-tetramethyl-l-piperidino- xyl free radical; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidin-l-oxyl free radical

0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00025-C

fluorescence substitution titrations using 4-methylumbel- liferyl a-D-mannopyranoside as the indicator ligand [5]. The kinetics of interaction of a- and /3-monosaccharides with Con A have been studied by fluorescence and spectral stopped-flow techniques [6-8].

The structure of Con A crystals of space group I222 has been solved by X-ray diffraction studies [9,10]. Carbon-13 and fluorine NMR relaxation measurements in aqueous solutions were used to calculate the distance of the ligands methyl a- and fl-o-glucosides and of N-trifluoroacetyl D-glucosamine from the S1 metal site of Con A and to estimate the binding orientation of the sugar [11-13]. Since the 1222 crystals of Con A dissolve on addition of saccharides, it was not possible to solve the structure of Con A-saccharide complexes until complexes of the lectin with some iododerivatives of glucose were crystallized in space group C2221:X-ray studies at 6 ,~ resolution con- firmed the location of the carbohydrate binding site found earlier by NMR. In 1989 [14] the structure of the Con A complex with methyl a-D-mannopyranoside crystallized in space group P2~ 2121 was reported.

We present here the results of proton NMR relaxation studies on complexes of Con A with a series of R-a-D-glu- cosides. Earlier attempts to calculate distances in these complexes from proton NMR data have given results incompatible with a3C- and 19F-NMR data [15]. The nov-

elty in the present work is that correlation times were obtained experimentally for more than one protons of the ligand; their use has led to meaningful distances and to some important indications on the influence of the aglycon on the sugar binding. In the ligands studied the sugar was mannose or glucose and R was methyl, 4-nitrophenyl, 4-methylumbelliferyl, 2,2,6,6-tetramethylpiperidinyl and TEMPO (Fig. 1).

2. Materials and methods

o-MUM

The synthesis of ct-PG and ot-TG was carried out according to Plessas and Goldstein [16] using slightly modified conditions [17]. The two glycosides were identi- fied by melting points, 1H-NMR (a-PG: H-1 (5.31 3), H-I' (4.42 8), H-2' (2.40 and 2.47 8) CH 3 (1.65 and 1.67

B), H-2' (2.40 and 2.47 8) CH 3 (1.65 and 1.67 8)) or X-ray crystallography (a-TG [18]). Con A was obtained from Sigma (Type IV, highly purified, lyophilized, water- soluble), methyl a-D-mannoside, p-nitrophenyl a-D-man- noside, p-nitrophenyl OL-D-glucoside, 4'-methylumbel- liferyl Ct-D-glucoside, and 4'-methylumbelliferyl a-D-man- noside from SERVA. All solvents and chemicals used in the synthesis and preparation of buffer solutions were analytical grade or purified by distillation and crystalliza- tion. The protein concentrations were measured by UV absorbance (E]8~ om= 12.4 at pH 5.6) [2]. Zn-Con A and Mn-Con A were prepared from native Con A according to Borrebaeck and Mattiason [3] with a slight modification in the technique of obtaining fragment-free Con A: the addi- tion of NH4HCO 3 (1%, w / v ) took place in the presence of NaC1, 1 M. In this way, the process of precipitation was slowed down resulting in better yields of intact protein.

o-HUG

0 o - P N G

~NO~

a-PG I,

H 'A

o-PNM

216 A. Troganis, C.L Stassinopoulou /Biochimica et Biophysica Acta 1206 (1994) 215-224

~CH~ o-TG

1

Fig. 1. Molecular structures of glycosides.

A. Troganis, C.L Stassinopoulou / Biochimica et Biophysica Acta 1206 (1994) 215-224 217

2.1. Fluorescence measurements

The binding constants were calculated from fluores- cence measurements using the substitution titrations method [5] on a Jasco FP 77 spectrofluorimeter at 295 K. Observed fluorescence intensities were corrected for dilution during the titration and for blanks. For each experimental point the cuvette was removed from the cavity, the appropriate amount of ligand solution was added and the samplewas thoroughly mixed by magnetic stirring. The magnet bar remained in the cuvette throughout the experiment.

The substitution titrations of Con A with c~-TG using a-MUM as the fluorescent indicator ligand were carried out by first adding protein to a solution of the fluorescent ligand and measuring the decrease in intensity. When no further decrease was observed, titration with the non-fluo- rescent ligand was started until the new equilibrium point was reached. The equilibria holding in the system are:

P + L = eL, K L = [PL] / [P] [L]

P + I = PI, K 1= [P l ] / [P ] [ I ]

where P denotes protein, L fluorescent indicator, I compet, ing ligand, and PL and PI the complexes of the protein with L and I, respectively. The fluorescence of the indiCa- tor (excitation 315 nm, emission 380 n m ) i s totally. quenched in the complex PL. The competing iigand and the complex PI do not fluoresce. The results were substi- tuted into the following equations to obtain the association constants K~ and KL:

1 1 - - +

F o - F F o - F = K L ( F o -Foo)Po

F o - F F o - F l o - t " o + - - L o +

F o - F= KL( F - Boo)

(1)

- + " P o - - L o ( 2 ) Kt KI F o - F F o - F ~

where Po, Lo, Io are the total concentrations of protein, fluorescent ligand and competing ligand; Fo, F and F= are the fluorescence intensities in the absence of protein, in the presence of protein and at infinite protein concentration (when all L is in the bound state), respectively. The latter, F~, is obtained from the y-axis intercept of Eq. (1).

2.2. Magnetic resonance methods and measurements

NMR measurements were carried on a Varian XL-100 FT and a Bruker AC200E spectrometers. A Bruker ER 200D-SRC spectrometer was used for the ESR spectra.

According to the simplified Solomon-Bloembergen equation [11-13], when the $1 site of the protein is occupied by a paramagnetic bivalent ion such as Mn 2+ the distances of the ligand nuclei from the ion can be calcu-

lated from Tlv, the paramagnetic contribution of the metal to T 1

1 B% 7 3 + (3)

Tip r 6 1 --[- (tOSTc) 2 1 -q- ( tOlTc) 2

where for Mn 2÷, r is the Mn2÷-proton distance, o) 1 the proton Larmor frequency, w s the electron Larmor fre- quency and % the correlation time of the magnetic dipolar interaction of the proton and the unpaired electron of the manganese ion. T w can be calculated from relaxation time measurements in Con A-glycoside complexes. Two sets of measurements of TloBs, the experimentally found relax- ation time, are needed: One in which the S1 site of Con A is occupied by the paramagnetic manganese ion and an- other with the zinc ion in the S1 site. Both Mn-Con A and Zn-Con A are fully active lectins. Tip is given by:

- - = - ( 4 )

TIp Mn Zn

TIM is obtained from the observed TI:

1 x , x b - - = + - - ( 5 ) TlOBS Tlf TIM -'1- T m

where Xf and X b to molar fractions of the free and bound ligand, Tlf and TiM the relaxation times of the free and complexed ligand and z m the lifetime of the complex.

The parameters needed to apply Eqs. (3)-(5), that is molar fractions, relaxation times, lifetimes of complexes and correlation times, were obtained experimentally.

2.3. Relaxation times T 1

T t measurements were carried in solutions of Con A in the presence of excess glycoside (15 to 20 times the concentration of protein). The inversion-recovery method ( 1 8 0 - t 1 - 9 0 ) was used. The T 1 observed values were calculated from the equation

M , = M = - ( M = - M o ) e -t/rl (6)

where M t is the intensity at time t, M= the intensity at equilibrium and M o the intensity at t = 0.

2.4. Lifetimes

The lifetimes, rm, of the Con A-glycoside complexes were obtained from temperature dependence studies of the ligand proton linewidths. The linewidth Av at half-height depends on T 2 and r m. It is known that as the temperature increases r m decreases, whereas T 2 and T 1 in liquids increase. The temperature dependence studies are carded in the presence of excess ligand. Under these conditions

Xb T2m + Zm = ~r( A v _ XfAvo) (7)

218 A. Troganis, C.1. Stassinopoulou / Biochimica et Biophysica Acta 1206 (1994) 215-224

where T2M is the transverse relaxation time of the ligand, Auo, Au the linewidth at half-height in the absence and the presence of Con A, respectively. A plot of 1og[Xb/Tr(Au --XfAuo)] vs. 1 /T gives the regions of low, intermediate and fast exchange.

2.5. Correlation times

The correlation times of Con A complexes with dia- magnetic glycosides were calculated from T 1 measure- ments at two frequencies, 100 MHz and 200 MHz using the relation derived from the Solomon-Bloembergen equa- tion:

Tip ( , o2 / /T ip to l ~---(1 ÷ Zc2tO2)/(1 + ~'~2to~) (8)

This relation holds provided % does not depend on frequency. The correlation time contains three terms: ~'R, the rotational correlation time, rs, the electron relaxation time and ~'m:

l / r c = 1//'T R -{- 1/~" s + 1 / ' r m (9)

The rotational correlation time and the lifetime of the complex are independent of frequency. It was found exper- imentally [15] that the electron relaxation time is also independent of frequency in the region 90-270 MHz.

The correlation time of TEMPO free radical in the Con A complex with a-TG was obtained from the relative intensities of the M ( - 1), M(0) and M(1) ESR peaks [19].

3. Results

3.1. Association constants

Table 1 shows the association constants, Ka, measured by fluorescence experiments. The values of K a for a-MM, ct-MG, a-PNG and a-MUM are, within experimental error, comparable to values given in the literature for these glycosides taking into consideration the differences in the temperature at which they were measured [6,7]. The asso- ciation constants of a-PG and a-TG are practically identi- cal. Since the piperidinyl and TEMPO groups are very

Table 1 Associat ion constants K a of ConA complexes at 295 K

Ligand K a ( x 104M - 1)

u -MUM 4.15 + 0.63 u -MUG a 0.41 ot-MM 0.49 + 0.32

a -PNM a 0.84 ot-PNG a 0.21

a- MG 0.17 :t: 0.03 a-PG 0.09 + 0.02

a -TG 0.13 5:0.04 TEMPOL - 0.02 5:0.02

a Ka for u-MUG, ot-PNM and a -PNG measured at 298 K are taken from

[6].

r"- A

2 -

t-- X )

<~

• I0"~

6

5

4

I/T(xlO3K -I)

Fig. 2. Plot of log [ X b / l r ( A u - XfAvo)] vs. 1 / T for CH 3 protons of

a -MUM. The straight line labelled r m gives the l ifetime of the complex at a given temperature.

close in structure, differing only at position 4 of the heterocyclic ring, it is expected that they would behave, as far as the lectinic interaction is concerned, in similar ways. We have shown previously that free TEMPOL quenches the fluorescence of Con A (excitation band at 295 nm, emmission at 340 nm) with an apparent association con- stant K ' = 9.2.103 M -1 [17]. Addition of a-MM does not perturb the TEMPOL-Con A interaction.TEMPOL does not displace a-MUM from the lectin sugar binding site. Substitution titration of the a-MUM-Con A complex with TEMPOL yielded a negative K a (Table 1) the absolute value of which is of the order of the experimental error. Consequently, a-TG has two modes of interaction with Con A, one as a glycoside (lectinic interaction) and a second as a nitroxide quenching non specifically the aro- matic fluorophores on the surface of the protein, not necessarily those situated in the vicinity of the sugar binding site.

3.2. Lifetimes of complexes

A plot of log (XJTr (Au - Xf Avo)) vs. 1/T is given in Fig. 2. The curve shows the dependence of the sum T2M + ~'m on temperature. In the region 274 to 295 K T2M does not change appreciably while the exchange rate goes from slow to intermediate. Therefore the change in the quantity A v - X f A v o reflects the change in the lifetime of the complex: the tangent to the experimental curve labelled ~'m is used to obtain ~'m at a given temperature. Above 295 K T2M starts increasing because the spin-spin relaxation process is slowed down and this accounts for the change in slope of the experimental curve.

At 293 K the lifetimes of the Con A complexes studied here are comparable: a mean value ~'m = 0.0015 s was used for the calculations of T1p.

A. Troganis, CL Stassinopoulou / Biochimica et Biophysica Acta 1206 (1994) 215-224 219

3.3. Times and distances f rom Mn 2 +

Correlation times were calculated for the ligand protons of Con A complexes with a-MUM, a-PNM and a-PG. Because of technical reasons measurements of T 1 in the complexes with a-PNG and a-MUG were performed only at one frequency (100 MHz). For the calculation of proton

distances in these complexes it was assumed that the correlation times of the corresponding mannosides(a-PNM and a-MUM) could be used. Tables 2 and Table 3 show the relaxation parameters obtained at 100 and 200 MHz, 293 K. The correlation times for ligand protons calculated from the above data are given in Table 4. Fig. 3 shows that accurate correlation times can be calculated only if the

Table 2 Relaxation times of ConA-a-D-mannosides. [ t~-MUM] = 5.73 • I0-3 M, [Zn-Con A] = 3 .92 .10-aM and [Mn-Con A] = 3.92.10-4 M. [ a-PNM] = 6.36. 10-3M

Ligand Proton 200 MHz 100 MHz

TIOBS TIM TIp T1OBS TIM Tip

a-MUM

a-MUM + Zn-Con A

a-MUM + Mn-Con A

a-PNM

a-PNM + Zn-Con A

ot-PNM + Mn-Con A

5' 0.62 5:0.01 6' 0.88 + 0.05 0.69 5- 0.03 8' 1.07 5- 0.04 0.71 5- 0.03 3' 2.42 ± 0.15 1.11 + 0.74 1 0.64 + 0.02 2 0.81 5:0.03 3 0.82 + 0.02

-CH 3 0.51 + 0.01 0.33 5- 0.02

5' 0.61 5:0.01 0.58 + 0.07 6' 0.87 5:0.02 0.73 5:0.08 0.67 __ 0.02 8' 1.04 _+ 0.01 0.70 + 0.05 0.69 _+ 0.02 3' 2.05 _+ 0.07 0.64 5:0.05 1.07 _+ 0.09 1 0.63 5:0.01 0.55 5:0.05 2 0.80 5:0.01 0.67 5:0.06 3 0.82 5- 0.04 0.80 5:0.08 -CH 3 0.48 5- 0.01 0.29 5:0.03 0.33 + 0.02

5' 0.56 _+ 0.02 0.24 5:0.03 0.42 + 0.06 6' 0.79 + 0.02 0.32 5:0.03 0.59 + 0.06 8' 0.89 + 0.02 0.25 5:0.05 0.40 5- 0.03 3' 1.29 -t- 0.04 0.16 5:0.01 0.21 ___ 0.01 1 0.59 + 0.01 0.27 5:0.02 0.52 ___ 0.07 2 0.71 5:0.02 0.25 5:0.02 0.42 5:0.03 3 0.72 + 0.04 0.25 5:0.03 0.37 5:0.02 -CH 3 0.43 5- 0.01 0.12 5:0.01 0.21 + 0.02

2' 2.03 + 0.08 3' 1.15 + 0.04 1 0.90 5- 0.02 2 1.09 5- 0.03 3 1.04 5- 0.03 6 1.02 5:0.06 5 0.61 5- 0.05

2' 1.48 + 0.06 3' 1.01 + 0.05 1 0.84 5- 0.01 2 0.97 + 0.03 3 0.99 5- 0.04 6 0.88 + 0.05 5 0.58 ± 0.05

0.27 5- 0.02 0.34 5:0.09 0.37 5- 0.03 0.32 5- 0.04 0.57 5:0.09 0.26 5- 0.04 0.32 5:0.05

2' 1.23 5- 0.02 0.15 + 0.01 0.38 5- 0.04 Y 0.89 + 0.01 0.18 5:0.01 0.38 5- 0.04 1 0.77 + 0.01 0.22 + 0.01 0.57 5- 0.05 2 0.83 5- 0.02 0.16 5- 0.01 0.31 5- 0.01 3 0.86 ± 0.04 0.21 + 0.04 0.34 ± 0.05 6 0.68 + 0.03 0.09 + 0.01 0.14 ± 0.02 5 0.53 5- 0.04 0.15 ± 0.03 0.30 + 0.03

0.53 + 0.02 0.50 + 0,02 0.71 _ 0,09

0.29 5- 0.02

1.44 + 0.08 0.86 5- 0.05

1.02 + 0.06 0.71 5- 0.05

0.68 5- 0.05 0.53 + 0.04

0.49 + 0.07 0.48 + 0.05 0.70 + 0.09

0.40 _ 0.06

0.11 _+ 0.01 0.08 5- 0.01 0.10 + 0.02

0.09 + 0.03

0.17 + 0.02 0.18 5- 0.04

0.06 + 0.01 0.06 5- 0.0l

0.14 __ 0.01 0.10 + 0.01 0.12 5- 0.02

0.12 + 0.02

0.09 + 0.01 0.09 + 0.01

220 A. Troganis, C.L Stassinopoulou / Biochimica et Biophysica Acta 1206 (1994) 215-224

ratio of T1p's corresponds to the ascending part of the curve T1p(2OO)/T1p(lO0) vs. r~. Accordingly, only a lower limit value is given in Table 5 for the correlation time of proton 2' of a-PNM for which the relaxation times ratio is larger than 4 making the uncertainty in ~'~ very important.

The values of ~'c range from 10 -8 to 10 -1 ° S. In previous NMR studies for the determination of ligand distance from the S1 site in Con A-glycoside complexes a single ~'c was used for all ligand nuclei [11-15]. As far as the pyrannose carbons are concerned this is a good approx-

Table 3 Relaxation times of ConA-a-D-glucosides

Ligand Proton 200MHz 100 MHz

TIOBS TIM TIp TIOBS TIM Tip

[ a-PG] = 3.67- 10- aM, [Zn-Con A] = 3.92.10-4M, [Mn-Con A] = 3.92.10-4M, 293 K

ot-PG 1' 0.51 5:0.02 1' 0.36 5:0.03 2' 0.22 5:0.01 2 0.23 5:0.01 -CH 3 0.36 ± 0.01 -CH 3 0.29 5:0.01

a-PG + Zn-Con A 1 0.44 5:0.01 0.16 5:0.01 1' 0.34 5:0.02 0.22 5:0.02 2' 0.23 5:0.01 0.24 5:0.02 2' 0.23 5:0.01 0.23 5:0.01 -CH 3 0.36 5:0.01 0.33 + 0.03 -CH 3 0.29 5:0.01 0.27 + 0.02

a-PG + Mn-Con A 1 0.41 5:0.01 0.11 _ 0.01 0.45 5:0.08 1' 0.29 ___ 0.01 0.07 5:0.01 0.11 + 0.01 2' 0.20 + 0.01 0.08 5:0.01 0.12 5:0.01 2' 0.20 5:0.01 0.08 + 0.01 0.12 5:0.01 -CH 3 0.33 5:0.01 0.16 + 0.01 0.32 5:0.04 -CH 3 0.27 5:0.01 0.13 _ 0.01 0.26 5:0.03

[a-MUG] = 2.12" 10- 3M, [Zn-Con A] = 1.36.10-4M, [Mn-Con A] = 1.32.10-4M, 300 K

a-MUG

0.26 + 0.02

a-MUG + Zn-Con A

0.25 + 0.01 0.18 + 0.03

0.23 5:0.01 0.08 5:0.01 0.14 + 0.03

5' 1.00 5:0.06 8' 1.57 5:0.10 3' 3.15 + 0.20 1 0.83 5:0.07 -CH 3 0.77 + 0.04

5' 0.94 + 0.07 8' 1.26 + 0.05 3' 2.39 5:0.15 1 0.79 + 0.05 -CH 3 0.72 5:0.03

a-MUG + Zn-Con A 5' 0.74 + 0.05 8' 0.81 + 0.06 3' 1.37 5:0.10 1 0.60 + 0.04 -CH 3 0.58 + 0.01

[ ~-PNG] = 6.31 • 10- 3M, [Zn-Con A] = 3.33 • 10-4M, [Mn-Con A] = 3.25 • 10-4M, 300 K

a-PNG 2' 2.08 5:0.09 3' 1.26 _+ 0.08

ot-PNG Zn-Con A 2' 1.56 + 0.09 3' 1.11 + 0.04

0.49 + 0.06 0.28 + 0.01 0.47 + 0.06 0.48 + 0.06 0.34 _ 0.04

0.11 + 0.02 0.07 + 0.01 0.11 + 0.01 0.09 + 0.01 0.09 + 0.01

0.25 + 0.04 0.31 + 0.05

0.15 + 0.02 0.10 + 0.01 0.14 + 0.01 0.11 + 0.01 0.13 + 0.01

a-PNG Mn-Con A 2' 0.91 + 0.03 0.06 + 0.01 0.08 + 0.01 y 0.75 + 0.02 0.07 + 0.01 0.09 -I- 0.01

A. Troganis, C.L Stassinopoulou /Biochimica et Biophysica Acta 1206 (1994) 215-224 221

O 3

(21-

O

(1_ I---

I-

10-11

8 '

3'

L;

r

0-a 10- r 1 0 - t 0 1 0 - 9

Tc(S) Fig. 3. Dependence of the T1p(2OO)/Tzv(lO0) ratio on the correlation time for protons 3' and 8' of the ConA-(a-MUM) complex. The error in calculating the correlation time of 8' is much larger than for 3' due to the shape of the curve.

imat ion : 13C T 1 m e a s u r e m e n t s h a v e y ie lded va l id dis-

t ances ve r i f i ed la ter by X - r a y resu l t s in the sol id s tate

[14,20,21]. The m e c h a n i s m s o f r e l axa t ion d i f fe r for c a r b o n

and h y d r o g e n nucle i . C a r b o n re l axes m a i n l y t h r o u g h the

d ipo la r in t e rac t ion w i t h the p r o t ons c o v a l e n t l y a t t ached to

it. In the case o f p r o t o n o the r m e c h a n i s m s are i m p o r t a n t

such as s p i n - r o t a t i o n a l in te rac t ion , s e g m e n t a l m o t i o n s etc.

Table 4 Correlation times, MHz

%, obtained from T 1 measurements at 100 and 200

Complex Proton Tlv(200)/ r e (s) T1p(100)

ConA-ot-MUM 6' 3.97 + 0.08 8' 3.88 _+ 0.08 3' 1.72 + 0.12 -CH 3 1.72_+0.13

(1.60 + 0.64). 10- 8 (7.94 + 2.26). 10- 9 (8.95 _ 1.00). 10- to (9.00_+ 1.11). 10 -1°

ConA-a-PNM 2' 4.16+0.19 > 1.60,10 -s 3' 3.97-1-0.10 (1.65___0.79).10 -8

ConA-ot-PG -C~'I 3 2.20 + 0.23 (1.30 4-__ 0.22)" 10 - 9

Therefore it is safer to use experimentally determined rc's to obtain distances from the Solomon-Bloembergen equa- tion. Our experimental results show that the ligand protons in each complex are characterized by individual correlation times reflecting their individual mobilities. Two groups of protons can be distinguished: In the first, ~-c is of the order of 10 -8 s, that is of the order of r R, the rotational correlation time of the protein-glycoside complex as ob- tained from fluorescence measurements [21], in the second, r c is of the order of 10 -1° to 10 -9 S. To the first group belong the aromatic protons of a-PNM, a-PNG and a- MUM; clearly those are more strongly bound to the pro- tein. To the second group belong the methyl group and hydrogen-3' of a-MUM and the piperidinyl protons of a-PG. The decrease in r~ of the a-MUM methyl protons

10 Gauss

r - -

Fig. 4. EPR spectra of a 1 • 10 -4 M solution of free ot-TG (outer trace) and of a solution of I • 10 -4 M a-TG in the presence of 2 • 10 - 4 Zn-Con A (inner trace). In both spectra pH 5.6, I = 0.8 (NaCI), acetate buffer. Instrument parameters: 0.79 mW microwave power, 9.43 GHz, 0.4 modulation amplitude.

222 A. Troganis, C.L Stassinopoulou /Biochimica et Biophysica Acta 1206 (1994) 215-224

must be due to the rotation of the methyl group; hydrogen-3' which is coupled to methyl ( J = 1.3 Hz) is probably relaxing through it [22]. In the case of a-PG the correla- tion time reflects the freedom of motion of the piperidinyl group as a whole.

For a-TG, where NMR T 1 measurements cannot be carried because of the presence of the free electron, free- dom of motion of TEMPO was detected by ESR. The ESR spectra of the Con A - a-TG complex are expected to be a superposition of spectra of the free and bound ligand in equilibrium. The spectrum of the free ligand consists of three sharp equidistant peaks. Protein-bound nitroxides give in general broadened unsymmetrical spectra due to the reduction in mobility of the free radical. Working with the complex of TEMPO-a-D-galactopyranoside and Griffo- nia simplicifolia I isolectin A4, which has a dissociation constant of 8 . 1 0 - 5 M , Goldstein et al. [23] were able to detect a decrease in the intensity of the free spectral component and the appearance of new broad lines due to the lectin-bound fraction. We obtained ESR spectra of a-TG in the presence of twofold excess protein. Under these conditions, according to the value of Ka, the associa- tion constant of the complex, 18% of the ligand should be bound. However, new lines attributable to immobilized TEMPO did not appear; there was only a broadening of the triplet lines (Fig. 4) implying that the correlation time of the bound nitroxide is much smaller than the rotational correlation time of the protein complex. From the relative intensities of the M ( - 1), M(0) and M(1) ESR peaks we estimated the correlation times of the bound nitroxide to be 1 .5 .10 -1° s, two orders of magnitude lower than r R of the protein complex. It appears that the TEMPO group is not interacting with the protein surface but is ' hanging' out of the binding site.

Table 5 gives distances, r, of ligand protons from the metal ion. All distances conform with the molecular geom- etry of the ligands and are in agreement with 13C and 19F-NMR data [11-15].

4. Discussion

Correlation times and distances obtained from NMR data are averaged over an ensemble of conformations [24]. In this respect they reflect more truly the actual situation in biological systems than the crystallographic data. Indeed, more than one crystal structures have been obtained for Con A and its complexes with simple sugars. This may be due to slight conformational changes of the binding site induced by cooperative effects of sugar substituents in general and, more precisely, by the nature of the aglycon in glycosides. Accordingly, one should not expect identical orientations for all mannosides or glucosides binding to Con A.

The only X-ray crystallographic study detailing the saccharide binding concerns the Con A complex to an

Table 5 Distances, r, of ligand protons from the S1 site in Con-A-(R-ot-D-glyco- side) complexes

Protons Tip (s) ~'c (s) r (,~) Frequency (MHz)

a-MUM 5' 0.42 2.0.10- s 15.06 _+ 1.82 200 6' 0.59 2.0.10- 8 15.89 + 2.05 8' 0.40 2.0.10- s 14.95 _+ 1.96 3' 0.21 9.0.10-10 20.44 _+ 0.92 1 0.52 5.0.10 -s 13.39 _+ 1.65 2 0.42 5.0.10 -8 12.90_+ 1.70 3 0.37 5.0.10-8 12.64 _+ 1.67 -CH 3 0.21 9.0.10 -1° 20.45_+1.31

ot-PNM 2' 0.38 2.0.10 - s 14.82 :t: 1.89 3' 0.39 2.0.10 -8 14.83_+1.89 1 0.57 5.0.10-8 13.57 _+ 1.78 2 0.31 5.0.10 -8 12.31 _+ 1.65 3 0.34 5.0.10-8 12.49 _+ 1.55 6 0.14 5.0.10 -s 10.83 _+ 1.31 5 0.30 5.0.10 -s 12.21 _+ 1.54

a-PG 1 0.45 5.0.10 -8 13.07-+ 1.53 -CH 3 0.32 6.6' 10-10 21.84_+ 1.81 -CH 3 0.26 6.6.10- l0 21.14_+ 1.57

a-PNG 2' 0.08 1.8.10- s 14.68 :i: 1.95 3' 0.09 1.8.10- s 14.93 -+ 1.98

a-MUG 5' 0.15 1.8.10- 8 16.34 + 2.03 8' 0.10 1.8.10 -8 15.15_+1.90 3' 0.14 8.8.10-10 20.82 5:1.46 1 0.11 1.8-10 -8 13.27-+1.61 -CH 3 0.13 8.0.10-~0 20.53 -+ 1.36

200

200

100

100

alkyl mannoside (a-MM) [14]. The a-anomeric substituent of carbon-1 is directed out of the site making no contact with the protein; no electron density is detected for the methyl group. Crystal structures for Con A complexes with aryl glycosides are not available. A study of the ability of recta-substituted alkylphenyl fl-o-glucopyranosides to in- hibit the binding of Con A to polysaccharides [25] has provided evidence for the existence of a relatively non-polar area adjacent to the specific carbohydrate binding site which could accommodate aromatic structures attached to the anomeric oxygen. The correlation between the inhibit- ing potencies of the arylglucosides and the Hammett elec- tronic substituent constant was poor indicating that 7r-rr charge-transfer bonding is not a dominant factor in deter- mining the binding t o Con A. It was suggested that interactions of aromatic aglycons with alkyl side chains may be responsible for the cooperative effects observed. The results on R-ot-o-glucosides and R-ot-o-mannosides presented here support this hypothesis. The binding con- stant K a (Table 1) is larger for R =-aryl, increasing for

A. Troganis, C.L Stassinopoulou /Biochimica et Biophysica Acta 1206 (1994) 215-224 223

glycosides from tetramethylpiperidinyl and tetrameth- ylpiperidinyloxyl to methyl, p-nitrophenyl and 4-methyl- umbelliferyl substituents in that order. A similar trend is observed in mannosides. In a model proposed for the binding of a-MG to Con A [26] the methyl group is pointing outside onto the surface of the protein in agree- ment with the X-ray results for a-MM. The correlation time and proton distance data for a-PG and a-TG given in Tables 4 and 5 also indicate that the alkyl aglycons are pointing to the surrounding solvent. Because of the size of the piperidinyl and TEMPO groups an additional factor of steric hindrance comes into play which seems to perturb the binding so that the association constants for a-PG and a-TG are less than for a-MG (Table 1). It is noteworthy that the correlation times of non-aromatic protons pre- sented here are of the same order of magnitude (10-1° to 10 -9 s) as those reported by Fuhr et al [15] for the methyl group of a-MG. However, the distances calculated from their data are not compatible with the position of the sugar binding site as determined from X-ray crystallography. They have assumed, for calculating distances, that outer sphere relaxation of the unbound ligand by the bound manganese ion makes an important contribution to Tlf. It turns out that this is not true.

Correlation times of glucoside aryl protons (a-PNG and a-MUG) are not available since we were not able to perform T 1 measurements at two frequencies. We can safely assume that they are of the same order to those measured for arylmannosides (Table 4). In arylmannosides the protons of aromatic aglycons have z c values compara- ble to the rotational correlation time of the protein molecules suggesting that they are firmly held to the lectin binding site. In a model which we have proposed earlier for the binding of a-PNM to Con A (Fig. 5), based on semi-empirical energy calculations and interactive graph- ics, we have indicated the possibility of some favorable interactions of the aglycon with the protein as, for exam-

ple, the formation of a hydrogen bond involving 07 of the NO 2 and OG1 of T226 [27]. This is in good agreement with the results of the inhibition studies. On the contrary, the orientation of the p-nitrophenyl group in our model is not compatible with the specific 7r-Tr interaction of the aromatic ring with the residues Y12 and Y100 of Con A proposed by Farina and Wilkins [6]. Fig. 4 shows that, in the model, the tyrosine residues are at the entrance of the sugar binding site, whereas the aromatic ring of a-PNM is lying on a groove of the protein with the NO 2 group near residue T226. Notice that the distances reported for the p-nitrophenyl protons of a-PNM in our earlier paper were r = 13.84 A for H-2' and r = 15.09 ,~ for H-Y vs. a mean value r = 14.83 ,~ for the aromatic protons given in the present paper. Although the difference is well within ex- perimental error we trust more the present result obtained on more modern equipment. The distances of the aromatic protons from the manganese ion reported in Table 5 imply that the same protein region might accommodate the aryl aglycons of both glucosides and mannosides.

Our results show that the Solomon-Bloembergen equa- tion can be applied to 1H-NMR relaxation data to obtain meaningful distances of ligand protons from protein para- magnetic centers provided the correct correlation times are used. Besides, the values of r c for these protons provide, per se, a measure of their interaction with the protein and can be used to estimate their involvement in cooperative effects on the sugar binding.

Acknowledgments

We wish to thank Dr. B. Petrouleas of the Institute of Materials Science for the recording of the ESR spectra.

Part of this work was presented in the doctoral thesis of A. Troganis (University of Athens, 1992).

Fig. 5. Stereo pair model of the ConA-(ot-PNM) complex in the mode of binding proposed in [23]. Some binding site residues are indicated.

224 A. Troganis, CL Stassinopoulou /Biochimica et Biophysica Acta 1206 (1994) 215-224

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