Proc. Nat. Acad. Sci. USAVol. 73, No. 2, pp. 322-326, February 1976Biochemistry

Magnetic resonance studies of concanavalin A: Location of thebinding site of a-methyl-D-mannopyranoside

(lectin/nuclear magnetic relaxation times/metal ion sites/non-polar site)

BRYAN J. FUHR, BRIAN H. BARBER, AND JEREMY P. CARVER*Departments of Medical Genetics and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Communicated by Elkan R. Blout, November 10, 1975

ABSTRACT The longitudinal nuclear magnetic relaxa-tion times of the methyl protons of a-methyl-D-mannopyra-noside have been measured at 90 MHz and 270 MHz in solu-tions of concanavalin A complexed with: (i) Mn2+ and Ca2+;and (ii) Zn2+ and Ca2+. Zn2+ and Mn2+ are known to bind insite SI and Ca2+ in site S2 of concanavalin A. Both sites mustbe occupied before monosaccharides will bind to the protein.In order to extract Tip, the paramagnetic contribution to thebound methyl relaxation time, from these observations therelaxation time of uncomplexed sugar was determined. FreeMn2+ contributed insignificantly to the latter value; how-ever, outer sphere relaxation was found to be large at boundMn2+ concentrations greater than 1 mM. Comparison of theresults obtained at the two frequencies allowed the determi-nation of the correlation time for the interaction between themethyl protons and the bound Mn2+ and the distance be-tween them (21.5 4 1.2 A). In contrast to previous resultsfrom nuclear magnetic resonance studies, this distance isconsistent with binding at the cavity proposed for the saccha-ride binding site by Becker et aL [J. Biol. Chem. 250, 1513(1975)], although it does not preclude possible binding siteson the surface of the molecule.

It is now firmly established that the plant lectin concanaval-in A (Con A) provides a valuable protein probe of cell sur-face structure and dynamics (1). An important feature of thebinding of Con A to cell surface macromolecules is the factthat the binding can be specifically reversed or prevented(2) by the addition of a molar excess of the monosaccharidesa-methyl-D-glucoside (a-MeGlc) or a-methyl-D-mannoside(a-MeMan). The specificity of this monosaccharide interac-tion with Con A has been studied in detail by Goldstein andcoworkers (3) and is illustrated by the fact that the structur-ally similar non-inhibitory monosaccharide a-methyl-D-ga-lactoside (a-MeGal) does not even bind to Con A. The bind-ing data suggest that there is only a single monosaccharideinhibitor site per intact monomer of Con A and the precisenature of this monosaccharide interaction with Con A is ofconsiderable interest in view of its potential relevance to afurther understanding of the way in which Con A interactswith a cell surface receptor (2). Cuatrecasas (2) has recentlypresented evidence indicating that the monosaccharide in-hibits Con A binding to the cell surface receptor of fat cellsin a noncompetitive fashion. This requires that separate sitesexist on the Con A structure for the monosaccharide inhibi-tor and the cell surface receptor. Consequently, an impor-tant aspect of defining the monosaccharide inhibitor interac-tion now becomes the determination of the site on Con A at

Abbreviations: Con A, concanavalin A; a-MeGlc, a-methyl-D-glu-copyranoside; a-MeMan, a-methyl-D-mannopyranoside; fl-IPG, ,-(o-iodophenyl)-D-glucopyranoside; a-MeGal, a-methyl-D-galacto-pyranoside; GalNAc; N-acetylgalactopyranoside; ESR, electron spinresonance.* Author to whom reprint requests should be addressed.

which these ligands bind. The high resolution x-ray crystalstructure of Con A has been published (4, 5), and it is knownfrom solution studies that there is a perturbation of the ConA structure upon the binding of a-MeMan (6, 7) as requiredby the Cuatrecasas proposal for the mechanism of inhibition.Clearly, the determination of the possible relationship be-tween this perturbation and the structural mechanism formonosaccharide inhibition would be facilitated by the local-ization of the site of interaction.

In this communication, an attempt is described to locatethe protein binding site for the methyl group of the most po-tent, nonderivatized monosaccharide inhibitor, a-MeMan,by measuring T1 values for the methyl group protons of a-MeMan in exchange with specific paramagnetic and dia-magnetic complexes of Con A. The relaxation times havebeen measured at two magnetic field strengths in order toremove any ambiguity concerning the correct value of thecorrelation time for the interaction. Considerable care hasalso been taken in correcting for the significant outer spherecontributions to the relaxation of the free sugar.

MATERIALS AND METHODS

The Con A used in this study was obtained from PharmaciaFine Chemicals (Batch no. 7623) and in all cases has beendemetallized (7), enriched in fragment-free molecules (8),and prepared in deuterated acetate buffer, 0.05 M sodiumacetate dg (Merck, Sharp and Dohme) in 0.2 M NaCl.

All spin-lattice relaxation times for the methyl protons ofa-MeMan were measured on a Varian XL-100, a BrukerWH-90, or a Bruker HX-270 spectrometer operating in theFourier transform mode at the ambient probe temperatureof 28 ± 20. The T1 values were determined by a nonlinearleast squares fit to the exponential variation of either thepeak height or the product of the peak height and the half-height linewidth following a 180'-r-90' pulse sequence as afunction of the pulse spacing T.The crystallographic distance measurements reported in

this study have been calculated from a set of refined coordi-nates for the tetrameric Con A:Mn2+:Ca2+ complex (9)which were generously provided prior to publication. Dr.Karl Hardman of the Argonne National Laboratory, Ar-gonne, Ill. also very kindly supplied coordinates prior topublication (5) which were used in the initial stages of thisstudy.

THEORETICAL

In a system for which a small molecule is in exchange be-tween a protein-bound site and free solution, the observedspin-lattice relaxation time, T1OBS, is described by theLuz-Meiboom expression (10).

322

BFProc.Nat. Acad. SCi. USA 73 (1976) 323

1 Xf Xb= +

T10BS T1f TiM + TM[1]

Xf and Xb are respectively, the mole fractions of the smallmolecule of interest free in solution and bound to the macro-molecule. Tif is the spin-lattice relaxation time for the nu-cleus in question during the time that it is not bound to theprotein and TiM the corresponding time for the bound mol-ecule. In this report, we are only concerned with the methylgroup protons for a-MeMan in exchange with the Con A:Mn2+:Ca2+ or Con A:Zn2+:Ca2+.The value of Tlf is usually determined by measuring the

relaxation time of the ligand nucleus in the absence of pro-tein. However, there are two potentially significant contri-butions to Tif which are not accounted for by this method.These contributions arise from the presence of free para-magnetic metal ions dissociating from the protein-metal ioncomplex and from outer sphere relaxation of the unbound li-gand by the bound paramagnetic metal ion. Measurement ofthe distance between the bound paramagnetic ion and a nu-cleus of the ligand requires accurate values of TiM as is de-scribed below. Clearly, errors in Tlf would affect the calcu-lated distances by causing errors in TiM. Thus careful esti-mation of the magnitude of the above contributions to Tif isrequired. The bound site relaxation time, TiM, for themethyl protons could potentially experience an additionalrelaxation mechanism from the interaction with the para-magnetic Mn2+ that would not be present in the interactionwith the Con A:Zn2+:Ca2+ complex. This additional relaxa-tion mechanism, the paramagnetic contribution to the relax-ation time, (Tip)Mn-1 can be described in this case as:

(TipP)Mn = (T1M)Mn1 - (T1M) Zn* [2]

The subscripts Mn and Zn represent the TiM values deter-mined for the paramagnetic and diamagnetic complexes, re-spectively. This paramagnetic contribution can then be re-lated to the interaction distance by the appropriate approxi-mation to the Solomon-Bloembergen expression (11, 12).

B T,(TLP)Mn = rMn6 1 + I2T2 [3]

In this expression the scalar interaction and terms in wSrTcare considered to be negligible. For the Mn2+-lH interac-tion the constant B = 8.61 X 10-31 rad erg sec-1 gauss-2. WIis the nuclear resonance frequency and rC is the correlationtime for the electron-nuclear dipolar interaction. (Tip)-1can be extracted from Eqs. 1 and 2, then the assignment ofTc (Eq. 3) enables one to calculate the Mn2+ to a-MeManmethyl proton interaction distance, rMn. By measuring(Tip)Mn-' at two values of WI, one can solve for both Tc andr.

RESULTS

Analysis of the factors influencing the Tlf values was ap-proached in three different ways. First, the free Mn2+ con-centrations in the solutions of Con A:Mn2+:Ca2+:a-MeManwere determined by electron spin resonance (ESR). Cohnand Townsend (13) showed several years ago that the ESRsignal from protein-bound Mn2+ is considerably less intenseand broader than that of the free Mn2+ aquo ion. As a result,it is possible to detect small amounts of free Mn2+ in solu-tions containing much larger concentrations of bound Mn2+

Table 1. Determination of outer sphere relaxation

T10BS (sec)*

Sample a-MeMan GalNAc ot-MeGal

1. 1.19 ± 0.03 1.32 ± 0.072. 0.47 ± 0.013. 0.45 ± 0.03 0.52 ± 0.04 -4. 0.54 ±0.04t5. 0.59 ± 0.016. 0.64 ±0.03-

The pD of the above samples was 5.4 in acetate buffer, 0.05 M so-dium acetate, and 0.2 M NaCl. The samples were: 1. Sugars in theabsence of protein or metals. 2. Con A 1.39 mM, Mn 1.33 mM, Ca12.4 mM, aMeMan 12.3 mM. 3. Con A 1.23 mM, Mn 1.18 mM, Ca11.0 mM, aMeMan 10.9 mM, GalNAc 11.7 mM. 4. Con A 1.39mM, Mn 1.33 mM, Ca 12.4 mM, aMeGal 11.9 mM. 5. Con A 1.09mM, Mn 1.04 mM, Ca 9.71 mM, aMeMan 129 mM, GalNAc 10.3mM. 6. Con A 0.86 mM, Mn 0.81 mM, Ca 7.52 mM, aMeMan 323mM, GalNAc 8.1 mM.* Obtained at 100 MHz.t 4.5% free Mn2+ by ESR.t 5% free Mn2+ by ESR.

by measuring the intensity of the sharp spectrum which ap-pears superimposed on the broad background of the boundMn2+ spectrum.

Before Ca2+ is added, about 17% of the total Mn2+ is freein solution (Con A 2.02 mM, Mn 2.04 mM, pD 5.2); how-ever, after addition of an excess of Ca2+ (13.2 mM) theamount of free Mn2+ falls to about 6%. This result is consis-tent with tighter binding of Mn2+ in site Si when Ca2+ oc-cupies site S2. Finally, when a-MeMan is added (12.1 mM),a small further decrease in free Mn2+ occurs (4% or 60,Mfree Mn2+), which might be due to the known conforma-tional change in Con A upon a-MeMan binding (15-17).Longitudinal relaxation time measurements of the methylprotons of aMeMan at these concentrations are unaffectedby Mn2+ concentrations of 100 ,M or less.

Initial attempts to determine the outer sphere contribu-tion were carried out by making T1 measurements on themethyl protons of two saccharides which have been indicat-ed not to bind to Con A (3), N-acetylgalactosamine (Gal-NAc) and a-MeGal. The results of measurements carried outat 100 MHz are shown in Table 1. The TI0BS values for freea-MeMan and GalNAc are between 1.2 and 1.3 sec; that fora-MeGal is expected to be similar. When a-MeMan is boundto the Con A:Mn2+:Ca2+ complex (sample 2) the T10Bs isconsiderably shorter than the free value due to dipole-di-pole interaction between Mn2+ and the methyl protons.Upon addition of GalNAc to sample 2 (resulting in sample 3)the T1 of a-MeMan remains unchanged and that of GalNAcis considerably shortened from its free value. A similar T1for a-MeGal in a solution of Con A:Mn2+:Ca2+ (sample 4)was obtained.From these results, assuming that the galactose sugars do

not bind to Con A, their shortened Tj's must result from anouter sphere contribution. The ESR spectrum of sample 4revealed only about 5% free Mn2+ present, which would notshorten the T1 of a-MeGal.To further test the possibility of significant outer sphere

contributions, 100- and 300-fold excesses of a-MeMan (overthe Con A concentration) were added to sample 4 (resultingin samples 5 and 6). Under these conditions less than 1% and0.3%, respectively, of the a-MeMan will be bound to Con A,assuming a KD of 80 MM for the Con A-a-MeMan interac-

Biochemistry: Fuhr et al.

Proc. Nat. Acad. Sci. USA 73 (1976)

- 7 -8 -9 -10log to (sec)

FIG. 1. A plot of the calculated distance from the Mn2+ site ofCon A to the bound a-MeMan methyl group as a function of TC(assuming a TM of 0.032 sec) for the frequencies indicated. Thearea of overlap in the error limits is hatched, while the area ofprobable values of rMn and TC is crosshatched (see discussion inthe text).

tion (14), and the main contribution to TIOBS must be due tothe Tlf term (Eq. 1). From the results shown in Table 1, it isagain seen that the TIOBS values are shorter than the freevalues. The slightly longer values for samples 5 and 6 com-

pared to those for samples 2 and 3 reflect the lower Mn2+concentrations in the former samples. It was also shown thatin Mn2+_a-MeMan solutions, the T, does not depend on thesugar concentration, only the Mn2+ concentration. Further-more, the ESR of sample 6 indicated only 5% free Mn2+, so

that free Mn2+ was not a source of the enhanced relaxation.The above results demonstrate that the outer sphere con-

tribution to Tif is quite significant. In fact, at Mn2+ concen-

trations greater than 1 mM it causes the free term in Eq. 1 todominate the expression, making measurements of TiM andrMn impossible. Therefore, in order to determine TiM valuesit was necessary to lower the Mn2+ concentration in order todecrease the significance of the outer sphere effect.The T0oBs values for the methyl proton resonance of a-

MeMan in the presence of the Con A:Mn2+:Ca2+ and ConA:Zn2+:Ca2+ complexes at the indicated concentrations are

listed in Table 2. The Mn2+ concentrations were about 0.6mM and the excess of Con A over Mn2+ ensured that negli-gible free Mn2+ was present in solution.

For the Mn2+ solutions an extrapolation of plots of (1/TiOBS) versus Xb to zero fraction bound yielded the valuesfor the corrected T1f's used in the calculations. These valueswere 0.93 and 1.08 sec, respectively, for the data at 90 and270 MHz. Note that the 90 MHz value of 0.93 sec is signifi-cantly less than that of 1.19 sec obtained for the free sugar

and reflects a significant outer sphere effect even at theselower Mn2+ concentrations.An example of the calculation of T1M,Mn for the 90 MHz

data is given below. From Eq. 1, inserting the appropriatevalues, one obtains:

1 0.82 0.18=+

0.54 0.93 TIM Mn + TM

The fraction of sugar bound is calculated as the ratio ofthe concentrations of Con A:Mn2+:Ca2+ to the total sugarconcentrations. Such a procedure is valid since a-MeMan

binds tightly to Con A [KD = 80 IAM (14)] and the method ofCunningham et al. (8) for enrichment in intact subunitsyields a preparation which binds one equivalent of saccha-ride per monomer (15). From this procedure one obtains avalue of 5.33 L 0.72 for (T1Mmn + TM)1'.The linewidth of the a-MeMan resonance was used to es-

tablish the limits expected for TM. The observed linewidth,AV1/2 = 2.3 4 0.3 Hz, is related to TIoBs by APv = (1/lrT2oBs). Assuming Tlf = T21 and using the Swift-Connickexpression (16) for the observed T2, the limits of TM are 0 <TM < 0.032 sec. This range of values should be compared tothe value of 0.025 sec determined for the a-MeGlc interac-tion with Con A (17, 18). The latter value should perhaps beconsidered the lower limit for a-MeMan in view of thegreater affinity of this sugar for Con A. Using each of thesethree values for TM (0, 25, and 32 msec), the correspondingvalues for (1/TIMMn), (1/TlpMn) and rMn were calculated.

In Fig. 1 is shown a plot of rMn, the distance from thebound Mn2+ to the average position of the bound a-MeManmethyl protons versus Ts, the correlation time for the Mn2+proton interaction, using the present data at both 90 and 270MHz and a TM of 0.032 sec. Similar plots were constructedfor TM values of 0 and 0.025 sec. Considering the standarddeviations in the least squares determination of TIOBS to bethe largest source of error, the errors in these plots are indi-cated. The possible range of rMn and TC values is indicatedin Fig. 1 by the hatched area. For reasons which will be ex-plained later, the lower Tc limit was obtained from thatpoint where the upper error curve for the 90 MHz datacrosses the upper curve for the 270 MHz data. A consider-ation of the plots obtained using the three TM values leads toa range of rMn values between 20.2 and 22.7 A and a rangeof Trc values between 2.5 X 10-10 and 9.4 X 10-10 sec.

DISCUSSION

The spin lattice relaxation times which have been measuredfor a-MeMan interacting with the Mn2+ complex of Con Aindicate that a-MeMan, when bound to Con A, experiencesa paramagnetic contribution to its relaxation time from theMn2+. The single Mn2+ binding site on Con A is well charac-terized by its observed position in the x-ray crystal structureof the Con A:Mn2+:Ca2+ complex (4, 5, 9).From the relaxation measurements presented in this

paper, a range of values for both rMn, the distance from thebound Mn2+ to the average position of the methyl protons ofthe Con A bound a-MeMan, and Tc, the correlation time forthe Mn2+_proton interaction, were simultaneously defined.This was possible since the measurements were carried outat two resonance frequencies, 90 and 270 MHz. Further-more, a significant outer sphere contribution to the T, of thefree a-MeMan in these solutions was found despite the useof lower concentrations of Mn2+ than in other nuclear mag-netic resonance studies (17-20). Presumably this effect isquite large relative to the bound contribution because of thelarge distance (>20 A) of the bound sugar from the Mn2+ inthe Con A complex. Free Mn2+ is not expected to contributeto the free sugar relaxation, under the conditions used to ob-tain the data given in Table 2, since negligible effects werefound at higher Mn2+ concentrations which would be ex-pected to have higher free Mn2+.

Using this corrected Tf value makes a significant differ-ence in the calculation of rMn and Tc. Therefore, in makingsuch calculations for paramagnetic complexes, it becomesimportant to check for outer sphere contributions.

324 Biochemistry: Fuhr et al.

Proc. Nat. Acad. Sci. USA 73 (1976) 325

Table 2. Con A:Mn2+:Ca2+ :ca.MeMan relaxation times

Ti0BS (sec)Xb 90 MHz 270 MHz

0.018* 0.86 ± 0.02 1.05 ± 0.030.093t 0.65 ± 0.04 0.91 ± 0.030.184t 0.54 ± 0.03 0.77 ± 0.020.290§ 0.83 ± 0.06 1.22 ± 0.05

pD of the above samples was 5.40 in acetate buffer, 0.05 M sodi-um acetate, and 0.1 M NaCl.* Con A 0.97 mM, Mn 0.64 mM, Ca 9.28 mM, aMeMan 35.3 mM.t Con A 0.82 mM, Mn 0.62 mM, Ca 8.96 mM, aMeMan 6.70 mM.t Con A 0.94 mM, Mn 0.64 mM, Ca 9.28 mM, aMeMan 3.47 mM.§ Con A 0.90 mM, Zn 1.14 mM, Ca 9.19 mM, aMeMan 3.43 mM.

The range of values obtained for Trc is worthy of com-ment. The expression for TC is given by:

1 1 1 1

Tc Tr TM T,

In this expression, Tr is the rotational correlation time for theparamagnetic metal ion-ligand interaction, TM is the life-time of the ligand bound to the protein, and TS is the elec-tronic spin relaxation time for the paramagnetic ion. Usingthe parameters determined in the previously reported fre-quency-dependence analysis of the relaxation enhancementproperties of the Con A:Mn2+:Ca2+ complex (7), the valuefor -T at 100 MHz is calculated to be 4.3 X 10-6 sec, and isindependent of frequency in the frequency range 90-270MHz. T. for dimeric Con A is estimated from depolarizationof fluorescence studies to be 1.8 X 10-8 sec (21) and a rea-sonable lower limit for TM is 2.5 X 10-2 sec (see above).These values indicate that Tc will be dominated by Ir. Thepresent data place an upper limit on Tc of 9.4 X 10-10 sec,more than an order of magnitude shorter than T. for dimer-ic Con A. However, Tr for the Mn2+ to methyl proton inter-action would be expected to be shorter than that of the pro-tein if local motion within the binding site or rotational mo-tion of the methoxy group significantly modulates the dipo-lar interaction with Mn2+. Because of the error limits in thepresent data, a lower limit cannot be placed on Tc. However,from the above discussion, it appears unlikely that Tc is sig-nificantly shorter than 10-10 sec. The point at which theupper error curves cross in the plot of Fig. 1 gives a lowerlimit of 2.5 X 10-10 sec for Tc.Brewer et al. (17, 18) first reported values for the distance

between Mn2+ at S1 and a bound monosaccharide. In thatstudy, it was necessary to choose between two possible valuesof Tc, 8 X 10-8 and 2 X 10-10 sec. The latter value was erro-neously reported as 2 X 10-9 sec in their work. The shorterTc value was rejected for a number of reasons and the valueof 8 X 10-8 sec was used in the distance calculations. Thisvalue is longer than the measured T. for dimeric Con A (21),suggesting that there is no motion of the a-MeGlc ring car-bons in the binding site.One of the reasons given by Brewer et al. (18) for reject-

ing the 2 X 10-10 sec value for Tc is that it predicts a T2mwhich is inconsistent with the observed linewidths. How-ever, calculation of the line width under these conditions re-quires an estimate of the chemical shift upon binding. Brew-er et al. (17) assumed it was negligible, since they observedno shift in the avMeGl carbon resonances in the presence of

2, we also observed no shift in the methyl proton resonance.

Considering once again the data of Table 2 at 100 MHz withXb = 0.18, Tif = 0.93 sec, we obtained a (T1MMn + TM) of0.19 sec. If TM is taken to be 25 msec (17) then Tc is 5 X

10-10 sec and T2MMn is 0.12 sec. With zero chemical shiftupon binding these parameters predict a linewidth of 0.7 Hzcompared to the observed value of 2.3 Hz. However, thereare exchange conditions under which the chemical shiftupon binding can contribute significantly to the linewidthand yet result in little alteration in the position of the reso-

nance (22). For the above conditions, it can be estimatedusing the Swift-Connick expression (16) for T2, that a mini-mum shift of 20 Hz in the methyl resonance upon bindingwould be required to produce a linewidth of 2.3 Hz. Thepredicted shift in the methyl resonance, however, wouldonly be 0.3 Hz. Under the experimental conditions used toobtain the data in Table 2, such a small shift would not bedetected.One consequence of free rotation of the a-methyl group

of a-MeMan in the bound site is that the distance rMn calcu-lated above will be an r-6-weighted average of the range

distances swept out during the motion. This effect is estimat-ed to introduce an uncertainty no greater than 0.5 A. Suchan uncertainty is well within the error limits for rMn andtherefore can be safely neglected.

In the 13C study of Brewer et al. (17, 18), referred toabove, the Tc value for the Mn2+ interaction with the ringcarbons was calculated on the basis of relaxation times mea-

sured for the interaction of the monosaccharide with theZn2+ complex of Con A. This Tc value then led to distancesfor the various carbons in the range 10.5-11.5 A. If one cal-culates the distances predicted by the data of Brewer et al.allowing both Tc and Tm to vary freely, maximum distancesof 14.8 A and 17.0 A can be obtained for C4 and CI corre-

sponding to Tm = 0 and Tc = 6 X 10-9 sec. Taking into ac-

count the C1 carbon to methyl distance of approximately 3A, it is clear that these distances are consistent with themethyl proton to Mn2+ distances determined in this study.An alternate explanation for the discrepancy between our

results and those of Brewer et al. could be the failure of thelatter workers to allow for the possible effects of free Mn2+or outer sphere effects on Ti1. Shorter values of Tlf lead tolonger predicted distances. In view of these considerations, itwould appear that the origin of the discrepancy between our

conclusions and those of Brewer et al. could lie either in theneglect of factors shortening Tlf or from the use of an incor-rect TC, or perhaps from both sources. It remains to be seen

whether or not these discrepancies will be resolved when the13C studies are repeated with appropriate controls for thepossible contributions to* Tif and a direct determination ofTc from the frequency dependence of Tip.

Crystallographic methods have led to the proposal of twodistinctly different binding sites as the one of importance forthe monosaccharide inhibitor binding to Con A. The first ofthese was advanced by Edelman and coworkers (15, 23)using the monosaccharide derivative f3-(o-iodophenyl)-D-glucopyranoside (W-IPG). The iodine atom of this monosac-

charide was located at the inner end of a relatively hydro-phobic cavity in the protein, approximately 15 A from thesurface. From this location the orientation of the saccharidemoiety in the site was inferred to be directed out of thepocket midway between the iodine atom and the surface ofthe protein. This direction afforded the greatest opportunityfor the numerous hydrogen-bonding contacts expected with

Con A. In the proton spectra used to obtain the data in Table

Biochemistry: Fuhr et al.

the protein.

Proc. Nat. Acad. Sci. USA 73 (1976)

Hardman and Ainsworth (24) have demonstrated that anumber of noninhibitory small molecules such as o-iodoani-line and o-iodophenol, as well as the noninhibitory saccha-ride derivative /3-(o-iodophenyl)-D-galactopyranoside alsobind to Con A with their iodine atoms in the position foundfor that of fl-IPG. This led them to propose that binding inthis site is determined by the iodophenyl substituent ratherthan the monosaccharide ring structure and does not, there-fore, reflect the location of the monosaccharide specific in-hibitor site on Con A. Their alternative proposal for the car-bohydrate specific site is a region on the monomer structureapproximately 12 A from the Mn2+ site and 7 A from theCa2 , in the vicinity of the side chains of Tyr 12 and Tyr101. This region has been proposed partly on the basis of itsinvolvement in a number of intermolecular contacts in thecrystalline lattice and the observation that the potent mono-saccharide inhibitors crack the Con A crystals when addedin sufficient concentration.The results of more recent solution studies carried out by

Becker et al. (15) led these authors to favor a single bindingregion in Con A, the cavity which contains a saccharide-spe-cific subsite adjacent to a hydrophobic binding subsite. Sincea single binding site per monomer is observed in solutionand since the non-polar noninhibitory compounds which, inthe crystal form, bind in the cavity do not appear to bind atall in solution, Becker et al. (15) raise the possibility that theinteractions between Con A and ,3-IPG may be different inthe two states. They suggest that in solution the saccharide-specific subsite is most significant but that upon crystalliza-tion a conformational change may enhance the bindingstrength of the hydrophobic subsite and reduce the influ-ence of the saccharide site.The range of Mn2+ to methyl proton distances obtained in

this study, 20.2-22.7 A, is consistent with a-MeMan bindingat the saccharide-specific subsite proposed by Becker et al.(15) if the methyl group is directed inwards towards the hy-drophobic subsite. However, under such circumstances thering carbons would have to be more distant from Mn2+ thanthe methyl protons (about 25 A). In the above discussion ofearlier 13C studies, it was pointed out that the use of shortercorrelation times extends the possible carbon to Mn2+ dis-tance into the range of 15-17 A. Such values are consistentwith our methyl proton distance, but only if the sugar mole-cule is oriented such that the methyl group is further fromS1. To obtain carbon distances of about 25 A, consistent withboth our methyl proton distance and the orientation re-quired for binding in the saccharide-specific subsite pro-posed by Becker et al. (15), would require that the neglectedcontributions to Tif in the 13C studies be almost as large asthe T1P observed. However, contributions of such magni-tude are ruled out by the observed (17) dependence of Tipon a-MeGlc concentration.On the other hand, methyl proton distances of 20-22 A

and carbon distances of 15-17 A are approximately 5 A toolarge for the saccharide site proposed by Hardman and Ains-worth (24). However, such distances are not inconsistent

with binding at other sites on the surface of the Con A mole-cule. Thus, considerably more data will be required beforethe precise location of the monosaccharide binding site ofCon A is known.

We thank Drs. W. Lutz and G. M. Edelman, Rockefeller Univer-sity, New York, for suggesting that we investigate the factors in-fluencing Tjf. We also thank Prof. Murray H. Freedman, Facultyof Pharmacy, University of Toronto, for the use of the Varian XL-100 and Dr. Alan Quirt for making T1 measurements during theinitial stages of this study. In addition, we are grateful to Prof.George C. Levy and Dr. Richard A. Komoroski, Florida State Uni-versity, for the measurements on the Bruker HX-270. Prof. BrianSykes, Harvard University, contributed many useful discussions.This research was supported by the Medical Research Council ofCanada through a Grant (MA-3732 to J.P.C.), a Studentship (toB.H.B.), and a Postdoctoral Fellowship (to B.J.F.)

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