Binding of 13 C - Enriched a - Methyl - D - Glucopyranoside to Concanavalin A as Studied by Carbon Magnetic Resonance ( carbon relaxation / metalloprotein / binding orientation )

Binding of a-methyl-D-glucopyranoside, uniformly enriched with 14% "IC, to zinc and manganese derivatives of concanavalin A at pH 5.6 was studied by pulsed Fourier transform carbon magnetic resonance techniques. Spin-lattice relaxation (T1) of the ["Clcarbons of the sugar was measured in the absence and presence of the two transition metal derivatives of the protein. In the presence of the manganese derivative of concanavalin A, selective relaxation of the sugar carbons was observed. The values for T1 reflect different distances between the carbons of the bound sugar and the manganese ion. Calculation of the distance between the manganese ion and each carbon of the sugar permit the 3-dimensional orientation of the bound sugar to be determined relative to the transition metal site in the protein. The results indicate that a-methyl-D-glucopyranoside binds to the protein in the Cl chair conformation with its 3and 4carbons closest to the manganese ion at a mean distance of 10 A. Concanavalin A (Con A), a hemagglutinating protein (1) isolated from jack beans (Canavalia ensiformi8), interacts with biopolymers containing appropriate carbohydrate residues in a manner resembling an antigen-antibody reaction (2). The protein binds monosaccharides with the D-glucopyranosyl configuration at the 3-, 4-, and 6-hydroxyl group positions, and interacts preferentially with the a-anomers of these sugars (3). The biological properties of Con A include selective agglutination of cells transformed by oncogenic viruses (4) and inhibition of tumor growth in experimental animals (5). The protomeric unit of Con A has a molecular weight of 27,000 (6, 7). The protein is a dimer between pH 3.5 and 5.6, and forms tetramers and other polymeric species at higher pH. Each protomeric unit has two binding sites for metal ions: a site for transition metal ions (SI) and a site specific for calcium ions (S2). The protein binds sugars only when both sites are occupied (8, 9). Con A derivatives in which Zn2+, C02+, or Mn2+ ions occupy the SI site possess similar capacities to bind sugars. The primary sequence and electron density map of Con A at 2resolution have recently been published (10). In this report, pulsed Fourier transform "IC nuclear magnetic resonance (NMR) was used to study the binding of IC-enriched a-methyl-D-glucopyranoside (a-MeDG) to the zinc and manganese derivatives of Con A. From these experiments, the distance from each carbon to the Mn2+ ion was Abbreviations: Con A, concanavalin A; a-MeDG, a-methyl->glucopyranoside; a-MeDM, a-methyl-D-mannopyranoside; NMR, nuclear magnetic resonance; NOE nuclear Overhauser enhancement. 1007 calculated, enabling the precise orientation of the bound sugar to be determined relative to the transition metal site in the protein. From the NMR data, the mean distance between the bound sugar and Mn2+ ion was calculated to be 10 A, which contrasts with the value of 20 A as proposed from x-ray crystallographic studies (10). MATERIALS AND METHODS a-MeDG uniformly labeled with 13.8% 'IC was synthesized from commercially available D-glucose uniformly labeled with 13.8% 'IC (Lot no. C-909, Merck, Sharp and Dohme). The method consisted of refluxing D-["C]glucose in methanol in the presence of a cation-exchange resin (11). The product, [1"C]a-MeDG, was isolated by separation on an anion-exchange column (12); elution was monitored by gas-liquid chromatographic analysis of the trimethylsilyl derivatives of the eluate. The product was recrystallized from ethanol-petroleum ether to a constant melting point, 167-169° (literature mp 166°) (13). The proton-decoupled "IC spectrum of [I"C]a-MeDG was in accord with literature assignments (14, 15). a-methyl-D-mannopyranoside (aMeDM) was obtained from Sigma. Zinc Con A (Zn-Con A) was obtained from Miles Yeda. Preparation of manganese Con A (Mn-Con A) will be reported elsewhere.t Both metal derivatives of Con A gave one band on polyacrylamide gel electrophoresis at pH 4.3 and were as active as "native" Con A in agglutinating sheep erythrocytes at room temperature. Atomic absorption analysis confirmed the presence of at least 90% of the appropriate transition metal in each protein sample, and two equivalents (± 1%) of transition metal ion per protein dimer. NMR sample solutions (2 ml) contained 47 mg/ml (0.87 mM) of the appropriate Con A sample in pH 5.60 0.1 N phosphate buffer. Ionic strength of the solution was brought to 1.0 by addition of KCl. Protein concentration was checked spectrophotometrically at pH 5.6, with an absorbance of 1.14 at 280 nm for 1 mg/ml of protein in solution (16). The ["8C]sugar was added to give final sample solutions containing 6.1-25 mM sugar. An internal coaxial tube containing deuterium oxide as the lock signal was used with 12-mm NMR sample tubes. Dioxane (10 al) was used as an internal 'IC standard. "IC NMR measurements were recorded at 23.4 kG on a modified Varian XL-100 pulsed Fourier transform spectrometer equipped with a Nicolet 1080 digital computer for signal accumulation and Fourier transformation t Brewer, C. F., Sternlicht, H., Marcus, D. & Grollman, A. P., manuscript in preparation. 1008 Biochemistry: Brewer et al. TABLE 1. Spin-lattice relaxation rates of ["C]a-MeDG in the presence of Znand Mn-Con A at 2£5 (Tm sec.) Concentration of protein was 0.87 mM = 0.025 4 0.005 Observed T, (sec) of Con A transition T, (see)* §, ¶ free sugar metal derivative Carbon ft = 0.071 ft = 0.14 T1m(sec) Tip(sec) (no protein) Zn-Con A 1 0.86 0.71 0.26 1.09 2 0.82 0.72 0.26 1.06 3 0.82 0.72 0.29 0.28§ 1.03 4 0.84 0.72 0.30 1.03 5 0.83 0.75 0.28 1.07 6 0.45 0.40 0.12 0.60 Mn-Con A 1 0.56 0.45 0.069 0.091 2 0.46 0.32 0.036 0.040 3 0.40 0.29 0.026 0.028 4 0.40 0.29 0.026 0.028 5 0.49 0.38 0.048 0.057 6 0.33 0.24 0.029 0.038 * T, rates were determined accurately to d0. 03 sec. t Total sugar concentration was 25 mM. $ Total sugar concentration was 12.5 mM. § An average TiM of about 0. 28 see for the ring carbons of a-MeDG bound to Zn-Con A was used in calculation of T1p. The difference in the individual TiM values reflect experimental errors in determination of T, values. I The estimated error in Tip is about 4 15% for the ring carbons and about t25% for the 6-carbon. (17). All NMR spectra were recorded with broad-band proton decoupling and 16K readout points. Spin-lattice relaxation times of the sugar carbons were measured by the 180'-At-90' two-pulse sequence method (18) by varying the time interval, At, between pulses. The T1 values were obtained from plots of log [2(1 M/Mo)-'] against At, where M is the net magnetization value of a given carbon along the direction of the external magnetic field (represented by the observed signal intensity) after the pulse interval following the 1800 pulse. Mo is the full magnetization value after 6T, times (complete relaxation) following the 1800 pulse. 2(1 M/Mo) -'is equal to 2.718 when At = Ti.