436
Biochimica et Biophysica Acta, 537 (1978) 436--445 © Elsevier/North-Holland Biomedical Press
BBA 38055 COBALT-CONCANAVALIN A AN INDEX OF INACTIVE AND ACTIVE CONFORMATIONAL STATES
A.D. CARDIN * and W.D. BEHNKE ** Department of Biological Chemistry, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267 (U.S.A.)
(Received May llth, 1978)
Summary Studies on the circular dichroic spectrum of cobalt-substituted concanavalin A have been continued in particular with respect to calcium- and saccharideinduced spectral perturbations reported previously (Kalb, A.J. and Pecht, I. {1973) Biochim. Biophys. Acta 303, 264--268; Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102,441--451}. We find that the addition of calcium or cadmium to (Co2÷)-concanavalin A induces slow time-dependent alterations in the extrinsic c o t t o n effects. Moreover, one equivalent of calcium is sufficient to cause maximal changes in the cobalt spectrum provided sufficient time is allowed for the effect to be observed. The addition of mono, di- and trisaccharides, specific for concanavalin A, have no resolvable effect upon the cobalt spectrum of concanavalin A (Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441--451). The data presented here suggest that these time-dependent processes are conformationally mediated and occur subsequent to $2 occupancy. Evidence is presented t h a t a particular calcium-cobalt-concanavalin A conformer exists which is responsible for the generation of activity in a lightscattering assay system.
Introduction Concanavalin A, a lectin isolated from the jack bean (Canavalia ensiformis), is able to bind to cell surfaces in a manner considered specific for certain saccharide acceptors of the glucopyranosyl and mannopyranosyl type configuration [3]. Concanavalin A and other lectins are known to possess both mito* I n P a r t i a l f u l f i l l m e n t f o r the d e g r e e o f D o c t o z o f P h i l o s o p h y . ** T o w h o m c o r r e s p o n d e n c e s h o u l d be addressed.
437 genic [4] and hemagglutinating activities [5,6]. Concanavalin A has been reported to restore density,dependent growth inhibition to transformed fibroblasts in tissue culture [7], Differential cytotoxicity towards normal and transformed cells has been reported for concanavalin A from in vitro studies, and inhibition of tumor development has been demonstrated in vivo [8]. The specific molecular nature of the interaction of concanavalin A with its membrane-bound receptors has remained largely conjectural due in part to an incomplete understanding of the structure-function relationships of concanavalin A itself. Concanavalin A is now known, from X-ray crystallographic and solution studies, to contain at least one monosaccharide binding site per protomeric unit [9--16], and at least two specific metal ion binding sites [11,17-21]. These sites, designated $1 and $2, are occupied by Mn 2+ and Ca 2+, respectively, in t h e native protein and appear to be necessary for concanavalin A activity [6,11]. As such, considerable attention has been focused upon the role of the $1 and S2 metals, and a number of biophysical investigations have been initiated in order to better understand the interaction of metals with concanavalin A [1,2,22--33,39]. By studying the cobalt spectral properties of (COX+)concanavalin A, it has been possible to detect multiple conformational forms in solution, and more importantly, provide evidence which strongly correlates calcium-induced conformational changes with substrate active and inactive states of the protein. The present data tentatively confirm results obtained by stopped-flow NMR [28] and magnetic field dispersion techniques [33] describing the interaction of the apoprotein with metals, and moreover, attests to the validity of previous observations via a route independent of magnetic resonance methods. Materials and Methods Concanavalin A was prepared according to the procedure of Agrawal and Goldstein [34] using jack bean meal purchased from Pfaltz and Bauer, Inc. The isolated protein was found to be homogeneous according to the criteria of sedimentation velocity, sedimentation equilibrium, discontinuous polyacrylamide gel electrophoresis and amino acid composition. With the buffer conditions employed (see figure legends), concanavalin A exists exclusively in its dimeric form with a molecular weight of 55 000. This was verified by meniscus-depletion ultracentrifugation techniques [35]. Apoconcanavalin A was prepared essentially according to the method of Brown et al. [33]. To 1.0 g protein in 30 ml distilled water, 1 M HC1 was added slowly with stirring to a final pH of 1.2. After 45 min at 25°C, the solution was transferred to thoroughly rinsed dialysis bags (which were previously boiled in a 5% NaHCO3/1 mM EDTA solution) and subjected to dialysis against four 6-1 changes of double glass-distilled water at 4°C. Various aliquots were then dialyzed exhaustively against the appropriate buffer system at 4°C. Protein concentrations were determined spectrophotometrically at pH 5.2 using an extinction coefficient ~o.1% ~2S0nmJ of 1.24 [33,36]. The analytical grade reagents used in preparing buffers were purchased from Mallinckrodt, Inc. Buffers were dithizone extracted to remove heavy metal ion contaminants and then subjected to spectrographic analyses. Further pre-
438 cautions against adventitious metal ion contamination were employed according to Thiers [37]. Stock solutions of cobalt, calcium, cadmium, and manganese were prepared approx. 0.1--0.2 M in double glass-distilled water and standardized by atomic absorption analyses. All metal salts were of spectrographic quality and were obtained from Apache Chemicals, Inc. A polysaccharide light-scattering assay was used similar to that described previously [2]. Assays were performed t w o ways: (i) At various times, following the addition of calcium to the cobalt-concanavalin A CD sample, aliquots were withdrawn and diluted to a final concentration of 1 mg/ml in 1.0 ml buffer containing 200 /~g/ml mannan (Sigma); and (ii) cobalt and calcium were simultaneously added to 1 ml solutions of apoconcanavalin A in buffer (1 mg/ml) and incubated for various time intervals. Polysaccharide precipitation was initiated b y the addition of 200/ag mannan. The assay was maintained at 25°C b y use of a constant temperature bath and the absorbance at 420 nm was monitored as a function of time with a Gilford-300 microsample spectrophotometer. Activity is based on the time to reach half-maximal velocity in the precipitation rate and henceforth denoted as T~ .A420. CD spectra were obtained at 25°C with a Cary 61 spectropolarimeter. Standardization of the instrument was accomplished using a 1 mg/ml aqueous solution of d-10~amphorsulfonic acid as specified by Varian Associates. Ellipticity, 8, is expressed as the observed ellipiticity in degrees. Cells with 1.0 cm pathlengths were utilized for all spectral measurements. Binding plots were cast in the form described originally b y Scatchard [38]. Results
As originally described by Kalb and Pecht [1] the substitution of cobalt for manganese in concanavalin A gives rise to a characteristic circular dichroic spectrum. Essentially three major bands are generated with maxima centered at 470 nm ([0] 470 nm = +420), 505 nm ([0] 505 nm = +360), and 530 nm ([0] 530 n m = +340). Moreover, the CD bands representative of the final (e.g. a spectrum which is unaffected b y further calcium addition or time factors) (Ca 2÷) (Co 2÷) concanavalin A spectrum (their Fig. 2) are represented b y maxima at 483 ([0] 483 n m = +193) and 505 nm ([0] 505 nm = +219). Fig. l a represents a time-dependent tracing of the cobalt spectrum of concanavalin A, monitored at 470 nm, after the addition of one equivalent of calcium. If calcium is added at time zero, an initial (Ca2*)-(Co2*)-concanavalin A spectrum is generated which is represented by a small instantaneous drop in the observed ellipticity followed b y a much slower time-dependent reduction in amplitude. It was found that the magnitude of this instantaneous reduction was directly proportional to the total a m o u n t of calcium added. Using this as an index of the a m o u n t of Ca 2÷ b o u n d to (Co2÷)-concanavalin A, a dissociation constant of 4.4 • 10 -3 M was obtained. Provided sufficient time is allowed following the addition of one equivalent of calcium, a final (Ca2÷)-(Co2÷)-concanava lin A spectrum is obtained in which no further fast or slow phase amplitude reductions are induced b y higher additions of calcium or b y saturating additions of specific carbohydrate substrates. At various times following the addition of calcium, the activity profile of the
439 0.60 _E0.75 0 0.70 I EQUIV. Ca ÷2
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80 ~
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0
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0.8 No 0.6' -20 .,=,~" 0.4"
i//
0.2
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x
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6
I0 14 MINUTES
18
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Fig. I . R e p r e s e n t a t i o n of the calcium-induced, time-dependent effects u p o n b o t h t h e s p e c t r a l p r o p e r t i e s o f b o u n d c o b a l t a n d t h e a c t i v i t y o f ( C o 2 + ) - ( C a 2 + ) - c o n e a n a v a l i n A. B u f f e r : 0.1 M s o d i u m a c e t a t e / 0 . 9 M NaCI ( p H 5.3). (a) [ ( C o 2 + ) - c o n e a n a v a l i n A ] = 9.9 • 10 -4 M, [Ca 2+] = 9.8 • 1 0 - 4 M, 25°C. (b) [(Co2+)-eon c a n a v a l i n A ] = 9.9 • 1 0 - 4 M, [Ca 2+] = 9.8 • 1 0 - 4 M, 50°C. F o l l o w i n g c a l c i u m a d d i t i o n , a l l q u o t s w e r e removed a t v a r i o u s t i m e intervals a n d a s s a y e d f o r a c t i v i t y . (c) [ a p o ] = 3.9 • 10 -5 M, [Co 2+] = 4 • 10 -5 M, [Ca 2+] = 4 • 10 -5 M, 25°C. S u p e r c r i p t s d e n o t e i n c u b e t i o n t i m e s w i t h c a l c i u m ( m i n ) p r i o r t o m a n n a n addition.
calcium-cobalt-concanavalin A complex was assayed directly from the CD cell as represented in Fig. lb. The longer the incubation time in the presence of calcium, the shorter the lag phase in activity. A spectrum-to-activity correlation is evident in Fig. lb. As progressive time-dependent reductions in the observed amplitude occur, a decrease in the time required to achieve a half-maximal rate, T~.A 42o, towards mannan precipitation also occurs. These phenomena, the slow phase amplitude reduction and the slow increase in competence towards mannan precipitability, are both initiated by the addition of calcium. Fig. lc illustrates that the length of the lag phase in activity varies with the metal incubation time. Additional experiments were carried out to further assess the effects of saccharides upon the cobalt CD spectrum of concanavalin A since differential effects were reported to occur [2]. As shown in Fig. 2a, if various saccharides specific for concanavalin A are added together with calcium, no observable net change occurs in the rate of the slow phase amplitude reduction, nor in the magnitude of the fast phase spectral reduction over those effects induced by calcium alone. Additionally, as shown in Fig. 2b, at subequivalent concentrations of calcium, addition of saccharides at intermediate time periods still do not produce observable effects. Moreover, subsequent inducible time-dependent changes are attributable only to the addition of further increments of calcium. In Fig. 3, a Scatchard plot was formulated describing the binding interaction of Ca2+ in the initial (Ca2+)-(Co2+)-concanavalin A complex. Calculations for each point were based on the value of the instantaneous amplitude reduction and each point on the graph represents a separate experiment performed in du-
440
.10 b o. {, °; :"
,08 o .@
°6I
Cd 2÷ > > Mn 2÷. The (COS÷)concanavalin A system in the absence of added calcium exhibits no spectral changes over a period of several days. Discussion The current b o d y of evidence presented here suggests that slow conformational events, which are triggered after the initial binding of calcium to cobaltconcanavalin A, lead to the increased competence for polysaccharide precipitation. Moreover, other ions such as Cd 2÷ and Mn 2÷ can produce the same effects b u t on a more protracted time scale. From these data, a minimum of two different conformers are postulated for the reconstituted protein; state A may represent a conformational state ineffectual in bringing a b o u t polysaccharide
442
0.76
0.74
-
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~O.7Z o.70-
X'~x"x'~'x",~ x I
I
0
Co 2"
ot/ 20'
I
5
I0 MINUTES
I
I 20
15
Cd2"
b
40
60
SO
I00
120
140
MINUTES
Fig. 5. E f f e c t of v a r i o u s m i x e d m e t a l p r e p a r a t i o n s o n b o t h the spectral p r o p e r t i e s of b o u n d c o b a l t and a c t i v i t y of ( C o 2 + ) - c o n c a n a v a l i n A. (a) [(Co2+)-concanavalin A] = 1 • 10 -3 M; z~--~--a Mn2+; o---o---o, Cd2+; X--×--X, Ca2+; 25°C. (b) [(Co2+)-concanavalin A] = 4 • 10 -S M. E a c h e x p e r i m e n t represents o n e e q u i v a l e n t of a d d e d m e t a l , 25°C. Buffer: 0.1 M s o d i u m a c e t a t e / 0 . 4 5 M N a N O 3 (pH 5.3) [ 2 0 ] .
precipitation whereas state B is competent to do so. Brown et al. [33] have presented evidence from magnetic field dispersion studies for the presence of 'unlocked' and 'locked' conformations of the (Ca2*)-(Mn2*)- and (Mn2*)(Mn2*)-concanavalin A complex. The conformational states A and B postulated here would presumably correspond to their 'unlocked' and 'locked' forms of concanavalin A, respectively. Grimaldi and Sykes [29] have presented a kinetic scheme deduced from stopped flow NMR studies which accommodates saccharide binding prior to the rate-limiting, calcium-induced conformational transition. Our data (Fig. lb) suggest that only conformational state B is capable of effecting the required activity needed for polysaccharide precipitation. Moreover, experiments utilizing 1'3Cd NMR indicate that the monosaccharide, a-me-
443 thyl-mannopyranoside, is n o t bound to the A form (or unlocked form) of the cadmium-concanavalin A complex [39]. That metals are b o u n d to the A form (unlocked) of the protein without a concomitantly recognized substrate binding function, suggests that the protein conformation, and n o t the presence of metals alone, is the primary factor in conferring saccharide binding activity to concanavalin A. The role of metal ions, as related to saccharide binding activity, appears to confer stability and hence maintenance of the proper conformation [39]. Moreover, there does appear to be a slight metal ion specificity involved in the degree of stability conferred to the final 'active' conformation [39]. Due to the calcium-induced time dependence observed in the (Co2+)-conca navalin A CD spectrum, a reevaluation of the saccharide and calcium binding interactions was undertaken [2]. First, the cobalt spectrum of concanavalin A appears insensitive to the addition of specific saccharide acceptors. It is possible that such saccharide-induced effects are n o t directionally transmitted to the S~ site or alternatively if they are, such environments a b o u t $1 remain relatively refractory to significant substrate-induced alterations. The latter explanation is favored in view of recent ~13Cd NMR results in which the S~ cadmium resonance is only slightly altered by monosaccharide binding whereas the $2 cadmium resonance is selectively removed from the spectrum [39]. Secondly, it is possible to separate the initial rapid binding event of calcium from the much slower calcium-induced conformational event. As such, a Scatchard plot can be formulated in which information concerning the strength of calcium binding as well as a lower limit to the number of calcium sites in the A state of the protein can be determined. It has been found (data n o t shown) that data cast in the form of a binding isotherm in which the time-dependent slow phase amplitude reduction makes a contribution to the observed ellipticity, results in a biased curvature [2]. The binding of Ca 2÷ to the A state can be described b y a relatively large dissociation constant, Kd ~ 4 . 10 -3 M. Moreover, the CD bands indicative of the A conformation are specifically altered by the addition of EDTA whereas the CD spectrum representative of the final (Ca2+)-(Co2÷)concanavalin A conformation remains insensitive to the addition of EDTA. With respect to the B conformation, cobalt remains inaccessible to EDTA (vida infra) and moreover, suggests a significantly decreased dissociation rate for Ca 2÷. Cobalt CD band assignments indicative of the time variant conformational forms described here have been made and will be presented elsewhere in detail (manuscript in preparation). These data suggest that Co 2÷ and Ca 2÷ are relatively free to exchange with the solvent lattice in the A conformation whereas in the B conformer, the metals would appear to be more tightly bound. Brown et al. [33] have shown that the binding of Mn 2÷ and Ca 2÷ in the 'unlocked' conformation is described b y rapid exchange and a large dissociation constant whereas the 'locked' form is characterized b y slow exchange with an off-rate on the order of many hours. Moreover, it appears from these data that the metal ion stoichiometries of both the A and B states are in the Ca 2÷ : Co 2÷ : concanavalin A ratio of 1 : 1 : 1. Finally, with respect to the cobalt system, it is evident that different ions capable of acting at $2, induce the conformational transition from A to B at different rates. This also appears to be the case for the (Ca2+)-(Mn2+)- and
444
(Mn2÷)-(Mn2+)-concanavalin A systems [33]. That the effects observed here can be ascribed to a specific conformational change is further warranted by similar time-dependent changes observed by us which occur in the near ultraviolet CD {310--250 nm). These transitions represent changes in the concanavalin A side chain chromophores (unpublished data). Brown et al. [33] have ascribed the 'unlocked' to 'locked' conformational transition to a cistrans isomerization of a peptide prolyl bond. This cis-trans isomerization is not evident in the far ultraviolet CD region, possibly due to the inherent insensitivity of this spectral region. Slow time-dependent effects in the near ultraviolet CD suggests that a secondary consequence of the cis-trans change may be an alteration in the concanavalin A side chain chromophores. Additional experiments, therefore, should include careful consideration of this region of the spectrum. Acknowledgements We would like to thank R.D. Brown, C.F. Brewer and S.H. Koenig for graciously supplying us with preprints and information of their work relevant to this manuscript. One of us, A.D.C., would like especially to acknowledge W.D. B. for his continued perseverance and guidance in this project. References 1 2 3 4 5 6 7 8 9 10 11 12
Kalb, A.J. and Pecht, I. (1973) Biochim. Biophys. Acta 303, 264--268 Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441--451 Goldstein, I.J., Hollerman, C.E. and Smith, E.E. (1965) Biochemistry 4 , 8 7 6 - - 8 8 3 Powell, A.E. ancL Leon, M.A. (1970) Exp. Cell Res. 62, 315--325 Edelman, G.M. and Millette~ C.F. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2436--2440 Inbar, M. and Sachs, L. (1969) Proc. Natl. Aead. Sci. U.S. 63, 1418--1425 Burger, M.M. and Noonan, K.D. (1970) Nature 228, 512--515 Shoham, J., Inbar, M. and Sachs, L. (1970) Nature 227, 1244--1246 Hardman, K.D. and Ainsworth, C.F. (1976) Biochemistry 15, 1120--1128 Becket, J.W., Reeke, Jr., G.N., Cunningham, B.A. and Edehnan, G.M. (1976) Natwre 2 5 9 , 4 0 6 - - 4 0 9 Kalh, A.J. and Levitzki, A. (1968) Biochem. J. 109, 669--672 Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P. (1973) Proc. Natl. Acad. Sci. U.S. 70,
13 14 15 16 17 18
Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P. (1973) Biochemistry 12, 4448--4457 Bessler, W., sharer, J.A. and Goldstein, I.J. (1974) J. Biol. Chem. 249, 2819--2822 Villafranca, J.J. and Viola, R.E. (1974) Arch. Biochem. Biophys. 160, 465--468 Alter, G.M. and Magnuson, J.A. (1974) Biochemistry 13, 4 0 3 8 - - 4 0 4 5 Weinzierl, J. and Kalb, A.J. (1971) FEBS Lett. 18, 268--270 Edelman, G.M., Cunningham, B.A., Peeke, Jr., G.N., Becker, J.W., Waxdal, M.J. and Wang, J.L. (1972) Proe. Natl. Acad. Sci. U.S. 69, 2 5 8 0 - - 2 5 8 4 Hardman, K.D. and Ainsworth, C.F. (1972) Biochemistry 11, 4910--4919 Shoham, M., Kalb, A.J. and Pecht, I. (1973) Biochemistry 12, 1914--1917 Becket, J.W., Reeke, Jr., G.N., Wang, J.L., Cunningham, B.A. and Edelman, G.M. (1975) J. Biol. Chem. 250, 1513--1524 MeCubbin, W.D., Oikawa, K. and Kay, C.M. (1971) Biochem. Biophys. Res. Commun. 43, 666---674 Koenig, S.H., Brown, R.D. and Brewer, C.F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 475--479 Sherry, A.D. and Cottam, G.L. (1973) Arch. Biochem. Biophys. 1 5 6 , 6 6 5 - - 6 7 2 Barber, B.H. and Carver, J.P. (1973) J. Biol. Chem. 248, 3 3 5 3 - - 3 3 5 5 Brewer, C.F., Marcus, D.M. and Grollman, A.P. (1974) J. Biol. Chem. 249, 4614--4616 Doyle, R.J., Thomasson, D.L., Gray, R.D. and Glew, R.H. (1975) FEBS Lett. 52, 185---187 Barber, B.H. and Carver, J.P. (1975) Can. J. Bioehem. 5 3 , 3 7 1 - - 3 7 9 Grimaldi, J.J. and Sykes, B.D. (1975) J. Biol. Chem. 250, 1618--1624 Doyle, R.J., Thomasson, D,L. and Nieholson, S.K. (1976) Carbohydr. Res. 46, 111--118
1007--1011
19 20 21 22 23 24 25 26 27 28 29 30
445 31 32 33 34 35 36 37 38 39
Alter, G.M., Pandolfino, E.R., Christie, D.J. and Magnuson, J.A. (1977) Biochemistry 16, 4034---4038 Carver, J.P., Barber, B.H. and Fuhr, B.J. (1977) J. Biol. Chem. 252, 3141--3146 Brown, III, R.D., Brewer, C.F. and Koeni_g, S.H. (1977) Biochemistry 16, 3883--3896 Agrawal, B.B.L. and Goldstein, I.J. (1967) Biochim. Biophys. Acta 147, 262--271 Yphantis, D.A. (1964) Biochemistry 3, 297--317 Yariv, J., Kalb, A.J. and Levitzki, A. (1968) Biochim. Biophys. Acta 165, 303--305 Thiers, R.E. (1957) Methods Biochem. Anal. 5, 273--335 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660--672 Bailey, D.B., Ellis, P.D., Cardin, A.D. and Behnke, W.D. (1978) J. Am. Chem. Soc., in the press
Biochimica et Biophysica Acta, 537 (1978) 436--445 © Elsevier/North-Holland Biomedical Press
BBA 38055 COBALT-CONCANAVALIN A AN INDEX OF INACTIVE AND ACTIVE CONFORMATIONAL STATES
A.D. CARDIN * and W.D. BEHNKE ** Department of Biological Chemistry, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267 (U.S.A.)
(Received May llth, 1978)
Summary Studies on the circular dichroic spectrum of cobalt-substituted concanavalin A have been continued in particular with respect to calcium- and saccharideinduced spectral perturbations reported previously (Kalb, A.J. and Pecht, I. {1973) Biochim. Biophys. Acta 303, 264--268; Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102,441--451}. We find that the addition of calcium or cadmium to (Co2÷)-concanavalin A induces slow time-dependent alterations in the extrinsic c o t t o n effects. Moreover, one equivalent of calcium is sufficient to cause maximal changes in the cobalt spectrum provided sufficient time is allowed for the effect to be observed. The addition of mono, di- and trisaccharides, specific for concanavalin A, have no resolvable effect upon the cobalt spectrum of concanavalin A (Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441--451). The data presented here suggest that these time-dependent processes are conformationally mediated and occur subsequent to $2 occupancy. Evidence is presented t h a t a particular calcium-cobalt-concanavalin A conformer exists which is responsible for the generation of activity in a lightscattering assay system.
Introduction Concanavalin A, a lectin isolated from the jack bean (Canavalia ensiformis), is able to bind to cell surfaces in a manner considered specific for certain saccharide acceptors of the glucopyranosyl and mannopyranosyl type configuration [3]. Concanavalin A and other lectins are known to possess both mito* I n P a r t i a l f u l f i l l m e n t f o r the d e g r e e o f D o c t o z o f P h i l o s o p h y . ** T o w h o m c o r r e s p o n d e n c e s h o u l d be addressed.
437 genic [4] and hemagglutinating activities [5,6]. Concanavalin A has been reported to restore density,dependent growth inhibition to transformed fibroblasts in tissue culture [7], Differential cytotoxicity towards normal and transformed cells has been reported for concanavalin A from in vitro studies, and inhibition of tumor development has been demonstrated in vivo [8]. The specific molecular nature of the interaction of concanavalin A with its membrane-bound receptors has remained largely conjectural due in part to an incomplete understanding of the structure-function relationships of concanavalin A itself. Concanavalin A is now known, from X-ray crystallographic and solution studies, to contain at least one monosaccharide binding site per protomeric unit [9--16], and at least two specific metal ion binding sites [11,17-21]. These sites, designated $1 and $2, are occupied by Mn 2+ and Ca 2+, respectively, in t h e native protein and appear to be necessary for concanavalin A activity [6,11]. As such, considerable attention has been focused upon the role of the $1 and S2 metals, and a number of biophysical investigations have been initiated in order to better understand the interaction of metals with concanavalin A [1,2,22--33,39]. By studying the cobalt spectral properties of (COX+)concanavalin A, it has been possible to detect multiple conformational forms in solution, and more importantly, provide evidence which strongly correlates calcium-induced conformational changes with substrate active and inactive states of the protein. The present data tentatively confirm results obtained by stopped-flow NMR [28] and magnetic field dispersion techniques [33] describing the interaction of the apoprotein with metals, and moreover, attests to the validity of previous observations via a route independent of magnetic resonance methods. Materials and Methods Concanavalin A was prepared according to the procedure of Agrawal and Goldstein [34] using jack bean meal purchased from Pfaltz and Bauer, Inc. The isolated protein was found to be homogeneous according to the criteria of sedimentation velocity, sedimentation equilibrium, discontinuous polyacrylamide gel electrophoresis and amino acid composition. With the buffer conditions employed (see figure legends), concanavalin A exists exclusively in its dimeric form with a molecular weight of 55 000. This was verified by meniscus-depletion ultracentrifugation techniques [35]. Apoconcanavalin A was prepared essentially according to the method of Brown et al. [33]. To 1.0 g protein in 30 ml distilled water, 1 M HC1 was added slowly with stirring to a final pH of 1.2. After 45 min at 25°C, the solution was transferred to thoroughly rinsed dialysis bags (which were previously boiled in a 5% NaHCO3/1 mM EDTA solution) and subjected to dialysis against four 6-1 changes of double glass-distilled water at 4°C. Various aliquots were then dialyzed exhaustively against the appropriate buffer system at 4°C. Protein concentrations were determined spectrophotometrically at pH 5.2 using an extinction coefficient ~o.1% ~2S0nmJ of 1.24 [33,36]. The analytical grade reagents used in preparing buffers were purchased from Mallinckrodt, Inc. Buffers were dithizone extracted to remove heavy metal ion contaminants and then subjected to spectrographic analyses. Further pre-
438 cautions against adventitious metal ion contamination were employed according to Thiers [37]. Stock solutions of cobalt, calcium, cadmium, and manganese were prepared approx. 0.1--0.2 M in double glass-distilled water and standardized by atomic absorption analyses. All metal salts were of spectrographic quality and were obtained from Apache Chemicals, Inc. A polysaccharide light-scattering assay was used similar to that described previously [2]. Assays were performed t w o ways: (i) At various times, following the addition of calcium to the cobalt-concanavalin A CD sample, aliquots were withdrawn and diluted to a final concentration of 1 mg/ml in 1.0 ml buffer containing 200 /~g/ml mannan (Sigma); and (ii) cobalt and calcium were simultaneously added to 1 ml solutions of apoconcanavalin A in buffer (1 mg/ml) and incubated for various time intervals. Polysaccharide precipitation was initiated b y the addition of 200/ag mannan. The assay was maintained at 25°C b y use of a constant temperature bath and the absorbance at 420 nm was monitored as a function of time with a Gilford-300 microsample spectrophotometer. Activity is based on the time to reach half-maximal velocity in the precipitation rate and henceforth denoted as T~ .A420. CD spectra were obtained at 25°C with a Cary 61 spectropolarimeter. Standardization of the instrument was accomplished using a 1 mg/ml aqueous solution of d-10~amphorsulfonic acid as specified by Varian Associates. Ellipticity, 8, is expressed as the observed ellipiticity in degrees. Cells with 1.0 cm pathlengths were utilized for all spectral measurements. Binding plots were cast in the form described originally b y Scatchard [38]. Results
As originally described by Kalb and Pecht [1] the substitution of cobalt for manganese in concanavalin A gives rise to a characteristic circular dichroic spectrum. Essentially three major bands are generated with maxima centered at 470 nm ([0] 470 nm = +420), 505 nm ([0] 505 nm = +360), and 530 nm ([0] 530 n m = +340). Moreover, the CD bands representative of the final (e.g. a spectrum which is unaffected b y further calcium addition or time factors) (Ca 2÷) (Co 2÷) concanavalin A spectrum (their Fig. 2) are represented b y maxima at 483 ([0] 483 n m = +193) and 505 nm ([0] 505 nm = +219). Fig. l a represents a time-dependent tracing of the cobalt spectrum of concanavalin A, monitored at 470 nm, after the addition of one equivalent of calcium. If calcium is added at time zero, an initial (Ca2*)-(Co2*)-concanavalin A spectrum is generated which is represented by a small instantaneous drop in the observed ellipticity followed b y a much slower time-dependent reduction in amplitude. It was found that the magnitude of this instantaneous reduction was directly proportional to the total a m o u n t of calcium added. Using this as an index of the a m o u n t of Ca 2÷ b o u n d to (Co2÷)-concanavalin A, a dissociation constant of 4.4 • 10 -3 M was obtained. Provided sufficient time is allowed following the addition of one equivalent of calcium, a final (Ca2÷)-(Co2÷)-concanava lin A spectrum is obtained in which no further fast or slow phase amplitude reductions are induced b y higher additions of calcium or b y saturating additions of specific carbohydrate substrates. At various times following the addition of calcium, the activity profile of the
439 0.60 _E0.75 0 0.70 I EQUIV. Ca ÷2
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Fig. I . R e p r e s e n t a t i o n of the calcium-induced, time-dependent effects u p o n b o t h t h e s p e c t r a l p r o p e r t i e s o f b o u n d c o b a l t a n d t h e a c t i v i t y o f ( C o 2 + ) - ( C a 2 + ) - c o n e a n a v a l i n A. B u f f e r : 0.1 M s o d i u m a c e t a t e / 0 . 9 M NaCI ( p H 5.3). (a) [ ( C o 2 + ) - c o n e a n a v a l i n A ] = 9.9 • 10 -4 M, [Ca 2+] = 9.8 • 1 0 - 4 M, 25°C. (b) [(Co2+)-eon c a n a v a l i n A ] = 9.9 • 1 0 - 4 M, [Ca 2+] = 9.8 • 1 0 - 4 M, 50°C. F o l l o w i n g c a l c i u m a d d i t i o n , a l l q u o t s w e r e removed a t v a r i o u s t i m e intervals a n d a s s a y e d f o r a c t i v i t y . (c) [ a p o ] = 3.9 • 10 -5 M, [Co 2+] = 4 • 10 -5 M, [Ca 2+] = 4 • 10 -5 M, 25°C. S u p e r c r i p t s d e n o t e i n c u b e t i o n t i m e s w i t h c a l c i u m ( m i n ) p r i o r t o m a n n a n addition.
calcium-cobalt-concanavalin A complex was assayed directly from the CD cell as represented in Fig. lb. The longer the incubation time in the presence of calcium, the shorter the lag phase in activity. A spectrum-to-activity correlation is evident in Fig. lb. As progressive time-dependent reductions in the observed amplitude occur, a decrease in the time required to achieve a half-maximal rate, T~.A 42o, towards mannan precipitation also occurs. These phenomena, the slow phase amplitude reduction and the slow increase in competence towards mannan precipitability, are both initiated by the addition of calcium. Fig. lc illustrates that the length of the lag phase in activity varies with the metal incubation time. Additional experiments were carried out to further assess the effects of saccharides upon the cobalt CD spectrum of concanavalin A since differential effects were reported to occur [2]. As shown in Fig. 2a, if various saccharides specific for concanavalin A are added together with calcium, no observable net change occurs in the rate of the slow phase amplitude reduction, nor in the magnitude of the fast phase spectral reduction over those effects induced by calcium alone. Additionally, as shown in Fig. 2b, at subequivalent concentrations of calcium, addition of saccharides at intermediate time periods still do not produce observable effects. Moreover, subsequent inducible time-dependent changes are attributable only to the addition of further increments of calcium. In Fig. 3, a Scatchard plot was formulated describing the binding interaction of Ca2+ in the initial (Ca2+)-(Co2+)-concanavalin A complex. Calculations for each point were based on the value of the instantaneous amplitude reduction and each point on the graph represents a separate experiment performed in du-
440
.10 b o. {, °; :"
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Cd 2÷ > > Mn 2÷. The (COS÷)concanavalin A system in the absence of added calcium exhibits no spectral changes over a period of several days. Discussion The current b o d y of evidence presented here suggests that slow conformational events, which are triggered after the initial binding of calcium to cobaltconcanavalin A, lead to the increased competence for polysaccharide precipitation. Moreover, other ions such as Cd 2÷ and Mn 2÷ can produce the same effects b u t on a more protracted time scale. From these data, a minimum of two different conformers are postulated for the reconstituted protein; state A may represent a conformational state ineffectual in bringing a b o u t polysaccharide
442
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Fig. 5. E f f e c t of v a r i o u s m i x e d m e t a l p r e p a r a t i o n s o n b o t h the spectral p r o p e r t i e s of b o u n d c o b a l t and a c t i v i t y of ( C o 2 + ) - c o n c a n a v a l i n A. (a) [(Co2+)-concanavalin A] = 1 • 10 -3 M; z~--~--a Mn2+; o---o---o, Cd2+; X--×--X, Ca2+; 25°C. (b) [(Co2+)-concanavalin A] = 4 • 10 -S M. E a c h e x p e r i m e n t represents o n e e q u i v a l e n t of a d d e d m e t a l , 25°C. Buffer: 0.1 M s o d i u m a c e t a t e / 0 . 4 5 M N a N O 3 (pH 5.3) [ 2 0 ] .
precipitation whereas state B is competent to do so. Brown et al. [33] have presented evidence from magnetic field dispersion studies for the presence of 'unlocked' and 'locked' conformations of the (Ca2*)-(Mn2*)- and (Mn2*)(Mn2*)-concanavalin A complex. The conformational states A and B postulated here would presumably correspond to their 'unlocked' and 'locked' forms of concanavalin A, respectively. Grimaldi and Sykes [29] have presented a kinetic scheme deduced from stopped flow NMR studies which accommodates saccharide binding prior to the rate-limiting, calcium-induced conformational transition. Our data (Fig. lb) suggest that only conformational state B is capable of effecting the required activity needed for polysaccharide precipitation. Moreover, experiments utilizing 1'3Cd NMR indicate that the monosaccharide, a-me-
443 thyl-mannopyranoside, is n o t bound to the A form (or unlocked form) of the cadmium-concanavalin A complex [39]. That metals are b o u n d to the A form (unlocked) of the protein without a concomitantly recognized substrate binding function, suggests that the protein conformation, and n o t the presence of metals alone, is the primary factor in conferring saccharide binding activity to concanavalin A. The role of metal ions, as related to saccharide binding activity, appears to confer stability and hence maintenance of the proper conformation [39]. Moreover, there does appear to be a slight metal ion specificity involved in the degree of stability conferred to the final 'active' conformation [39]. Due to the calcium-induced time dependence observed in the (Co2+)-conca navalin A CD spectrum, a reevaluation of the saccharide and calcium binding interactions was undertaken [2]. First, the cobalt spectrum of concanavalin A appears insensitive to the addition of specific saccharide acceptors. It is possible that such saccharide-induced effects are n o t directionally transmitted to the S~ site or alternatively if they are, such environments a b o u t $1 remain relatively refractory to significant substrate-induced alterations. The latter explanation is favored in view of recent ~13Cd NMR results in which the S~ cadmium resonance is only slightly altered by monosaccharide binding whereas the $2 cadmium resonance is selectively removed from the spectrum [39]. Secondly, it is possible to separate the initial rapid binding event of calcium from the much slower calcium-induced conformational event. As such, a Scatchard plot can be formulated in which information concerning the strength of calcium binding as well as a lower limit to the number of calcium sites in the A state of the protein can be determined. It has been found (data n o t shown) that data cast in the form of a binding isotherm in which the time-dependent slow phase amplitude reduction makes a contribution to the observed ellipticity, results in a biased curvature [2]. The binding of Ca 2÷ to the A state can be described b y a relatively large dissociation constant, Kd ~ 4 . 10 -3 M. Moreover, the CD bands indicative of the A conformation are specifically altered by the addition of EDTA whereas the CD spectrum representative of the final (Ca2+)-(Co2÷)concanavalin A conformation remains insensitive to the addition of EDTA. With respect to the B conformation, cobalt remains inaccessible to EDTA (vida infra) and moreover, suggests a significantly decreased dissociation rate for Ca 2÷. Cobalt CD band assignments indicative of the time variant conformational forms described here have been made and will be presented elsewhere in detail (manuscript in preparation). These data suggest that Co 2÷ and Ca 2÷ are relatively free to exchange with the solvent lattice in the A conformation whereas in the B conformer, the metals would appear to be more tightly bound. Brown et al. [33] have shown that the binding of Mn 2÷ and Ca 2÷ in the 'unlocked' conformation is described b y rapid exchange and a large dissociation constant whereas the 'locked' form is characterized b y slow exchange with an off-rate on the order of many hours. Moreover, it appears from these data that the metal ion stoichiometries of both the A and B states are in the Ca 2÷ : Co 2÷ : concanavalin A ratio of 1 : 1 : 1. Finally, with respect to the cobalt system, it is evident that different ions capable of acting at $2, induce the conformational transition from A to B at different rates. This also appears to be the case for the (Ca2+)-(Mn2+)- and
444
(Mn2÷)-(Mn2+)-concanavalin A systems [33]. That the effects observed here can be ascribed to a specific conformational change is further warranted by similar time-dependent changes observed by us which occur in the near ultraviolet CD {310--250 nm). These transitions represent changes in the concanavalin A side chain chromophores (unpublished data). Brown et al. [33] have ascribed the 'unlocked' to 'locked' conformational transition to a cistrans isomerization of a peptide prolyl bond. This cis-trans isomerization is not evident in the far ultraviolet CD region, possibly due to the inherent insensitivity of this spectral region. Slow time-dependent effects in the near ultraviolet CD suggests that a secondary consequence of the cis-trans change may be an alteration in the concanavalin A side chain chromophores. Additional experiments, therefore, should include careful consideration of this region of the spectrum. Acknowledgements We would like to thank R.D. Brown, C.F. Brewer and S.H. Koenig for graciously supplying us with preprints and information of their work relevant to this manuscript. One of us, A.D.C., would like especially to acknowledge W.D. B. for his continued perseverance and guidance in this project. References 1 2 3 4 5 6 7 8 9 10 11 12
Kalb, A.J. and Pecht, I. (1973) Biochim. Biophys. Acta 303, 264--268 Richardson, C.E. and Behnke, W.D. (1976) J. Mol. Biol. 102, 441--451 Goldstein, I.J., Hollerman, C.E. and Smith, E.E. (1965) Biochemistry 4 , 8 7 6 - - 8 8 3 Powell, A.E. ancL Leon, M.A. (1970) Exp. Cell Res. 62, 315--325 Edelman, G.M. and Millette~ C.F. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2436--2440 Inbar, M. and Sachs, L. (1969) Proc. Natl. Aead. Sci. U.S. 63, 1418--1425 Burger, M.M. and Noonan, K.D. (1970) Nature 228, 512--515 Shoham, J., Inbar, M. and Sachs, L. (1970) Nature 227, 1244--1246 Hardman, K.D. and Ainsworth, C.F. (1976) Biochemistry 15, 1120--1128 Becket, J.W., Reeke, Jr., G.N., Cunningham, B.A. and Edehnan, G.M. (1976) Natwre 2 5 9 , 4 0 6 - - 4 0 9 Kalh, A.J. and Levitzki, A. (1968) Biochem. J. 109, 669--672 Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P. (1973) Proc. Natl. Acad. Sci. U.S. 70,
13 14 15 16 17 18
Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P. (1973) Biochemistry 12, 4448--4457 Bessler, W., sharer, J.A. and Goldstein, I.J. (1974) J. Biol. Chem. 249, 2819--2822 Villafranca, J.J. and Viola, R.E. (1974) Arch. Biochem. Biophys. 160, 465--468 Alter, G.M. and Magnuson, J.A. (1974) Biochemistry 13, 4 0 3 8 - - 4 0 4 5 Weinzierl, J. and Kalb, A.J. (1971) FEBS Lett. 18, 268--270 Edelman, G.M., Cunningham, B.A., Peeke, Jr., G.N., Becker, J.W., Waxdal, M.J. and Wang, J.L. (1972) Proe. Natl. Acad. Sci. U.S. 69, 2 5 8 0 - - 2 5 8 4 Hardman, K.D. and Ainsworth, C.F. (1972) Biochemistry 11, 4910--4919 Shoham, M., Kalb, A.J. and Pecht, I. (1973) Biochemistry 12, 1914--1917 Becket, J.W., Reeke, Jr., G.N., Wang, J.L., Cunningham, B.A. and Edelman, G.M. (1975) J. Biol. Chem. 250, 1513--1524 MeCubbin, W.D., Oikawa, K. and Kay, C.M. (1971) Biochem. Biophys. Res. Commun. 43, 666---674 Koenig, S.H., Brown, R.D. and Brewer, C.F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 475--479 Sherry, A.D. and Cottam, G.L. (1973) Arch. Biochem. Biophys. 1 5 6 , 6 6 5 - - 6 7 2 Barber, B.H. and Carver, J.P. (1973) J. Biol. Chem. 248, 3 3 5 3 - - 3 3 5 5 Brewer, C.F., Marcus, D.M. and Grollman, A.P. (1974) J. Biol. Chem. 249, 4614--4616 Doyle, R.J., Thomasson, D.L., Gray, R.D. and Glew, R.H. (1975) FEBS Lett. 52, 185---187 Barber, B.H. and Carver, J.P. (1975) Can. J. Bioehem. 5 3 , 3 7 1 - - 3 7 9 Grimaldi, J.J. and Sykes, B.D. (1975) J. Biol. Chem. 250, 1618--1624 Doyle, R.J., Thomasson, D,L. and Nieholson, S.K. (1976) Carbohydr. Res. 46, 111--118
1007--1011
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Alter, G.M., Pandolfino, E.R., Christie, D.J. and Magnuson, J.A. (1977) Biochemistry 16, 4034---4038 Carver, J.P., Barber, B.H. and Fuhr, B.J. (1977) J. Biol. Chem. 252, 3141--3146 Brown, III, R.D., Brewer, C.F. and Koeni_g, S.H. (1977) Biochemistry 16, 3883--3896 Agrawal, B.B.L. and Goldstein, I.J. (1967) Biochim. Biophys. Acta 147, 262--271 Yphantis, D.A. (1964) Biochemistry 3, 297--317 Yariv, J., Kalb, A.J. and Levitzki, A. (1968) Biochim. Biophys. Acta 165, 303--305 Thiers, R.E. (1957) Methods Biochem. Anal. 5, 273--335 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660--672 Bailey, D.B., Ellis, P.D., Cardin, A.D. and Behnke, W.D. (1978) J. Am. Chem. Soc., in the press
