Small-angle X-ray scattering study of lysine transfer RNA ligase from yeast

J. Mol. Bid. (1973) 77, 153-15s

mall-angle X-ray Scattering Study of Lysine Transfer RNA Ligase from Yeast RAGNAR OSTERBERCJ, Bo SJ~BERG, LARS RYMO ANIS ULF LAGERKTWT

Department of Medicul Biochemistry of GGteborg,GGteborg,Xweden

University

(Received 29 November 1972) The scattered X-ray intensities from dilute solutions of Iysine transfer RNA ligase, in 0.1 x-phosphate buffer at pH 7.0, have been measured at 21”. The radius of gyration R (37.5 d), the molecular weight M (114,000), and the volume V (295,000 A3) were determined. a comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes

,P = 62~7,B = 50.1 and C = 23.5 A.

1. Introduction The elucidation

of the structural

basis for the specific interactions

between

a protein

and a nucleic acid is a major objective in molecular biology. The recognition by a ligasa? of its transfer RNA substrate is a good example of this general problem and has consequently received much attention. Nevertheless, we are still very far from a full understanding of the mechanism involved in the recognition procedure, and it is obvious that tllis will eventually require a thorough knowledge of the structure of both enzyme and tRNA. While the primary structure of a number of tRNAs is now known, and X-ray analysis of several tRNAs is under way in many laboratories, no information on the primary structure and conformation of any of the ligases is presently available. The reason for this is that structural studies of these enzymes have been seriously hampered by the difficulties of obtaining crystals suitable for crvst~allographic analysis. The crystallization of the 1ysine:tRNA ligase from yeast Y [Rymo et al., 1970; Lagerkvist et al., 1972) has been reported from this laboratory, and recently very promising crystals of 1eucine:tRNA ligase from the same source have been obtained in Fresco’s laboratory (Chirikjian et al., 1972). Wailer et al. (1974) have described crystals of an enzymically active major fragment of the methionine:tRNA ligase from Escherichia coli that also looks very promising for X-ray work. At the same time, in spite of considerable efforts in several laboratories, it has not been possible to crystallize a ligase with its tRNA substrate. It seemed important, therefore, to explore other methods that could give structural information on the ligase-tRNA system. As part of a small-angle X-ray scattering study of ligases and t The word ligase is used in this paper to denote an amino acid;tRNA ligase, 153

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ET AL.

their interactions with tRNA, this communication reports molecular parameters for the 1ysine:tRNA ligase from yeast. This enzyme has been well characterized with regard to molecular weight, amino acid composition, subunit structure and binding properties (Rymo et al., 1972), and an X-ray crystallographic analysis of it is now in progress in this laboratory.

2. Materials and Methods (a) Prepcbration of enzyme Lysine:tRNA ligase from Saccharomyces cerevisiae C836 was prepared as described previously (Rymo et al., 1970). Protein concentrations were calculated from protein nitrogen (Rymo et al., 1972), which was analysed either by a micro-Kjeldahl procedure (Strid, 1961) or by a micro-Dumas method (Kirsten, 1971). The enzyme samples used in the X-ray measurements were diluted from enzyme solutions that had been exhaustively dialysed against large volumes of 0.1 M-potassium phosphate buffer, pH 7.0. The dialysis buffer was used for the dilutions and for the recording of background scattering. (b) X-ray meawrements The X-ray small-angle scattering camera used in this study was that described by Kratky (Kratky, 1958; Kratky & Skala, 1958). It was equipped with an electronically programmed step scanning device (Leopold, 1968). The enzyme was found to be sensitive to X-rays when exposed to prolonged irradiation. However, during a period of 45 mm no changes were recorded as far as the X-ray scattering data were concerned. A special flow cuvette was therefore constructed, which ensured that a fresh solution would be introduced every 30 min during the measurements. Monochromatization was achieved by using a nickel fl filter and a pulse height discriminator in conjunction with a proportional counter. Scattering curves were recorded at 21.0” for 3 concentrations : 8.35, 4.0 and 2.3 mg/cm3 up to a scattering angIe, 28, of 0.059 radians. For every point on the curve, 4 x lo4 pulses were recorded. The scattering of the buffer solution was subtracted from each scattering curve. The experimental scattering curves were corrected for the length collimation effect by using a computer program (Heine, 1963) and an IBM 360/65 computer. This procedure, which involves an integration, was controlled as far as the tail end is concerned by generating intensity data in the range 28 = O-059 to 0.08 radians. The uncorrected intensity curve is proportional to l/(2@” for 28 > 0.030 radians. 3.

Results

(a) Radius of gyration When the logarithms of the desmeared normalized intensities were plotted against (2Bj2, the data at low values of 0 could be fitted to straight lines (Pig. 1). Thus, the slopes of these Guinier plots yielded the radius of gyration, R. After extrapolation to zero concentration, we obtained R, = 37.5 d (Table 1). Before slit correction the gyration radius, R,, was determined to be 37.7 8.

The molecuIar

weight,

(b) iWolecular weight a& volume M = 114,000, was calculated from the following

M =

K x a2 x IO/PO (21 - u&y x d x c ’

equation (1)

where 1,/P, is the absolute scattered intensity, 6, the parCal specific volume, K a constant (K = 21.0), a the distance between the sample and the plane of registration

X-RAY

SCATTERING

OF LYSINE-tRNA

1%6

3.0

2V (28)=x10*

I.0

LIGASE

FIN. 1. Guinier plots of intensity curves, corrected for the coilimation effect and obtained with solutions of lysine-tRNA ligase in 0.1 M-pOtaSSiURI phosphate buffer, pH 9.0, with the follow@ protein concentrations: (1) 8.3 mg/cm3; (2) 4.0 mg/onP; (3) 2.3 mg/om3. For clear presentation the ourves have been shifted 0.5 logarithmic unit on the ordinate.

(20.5 cm), rl the thickness of the sample (0.0454 cm), c the concentration of the sample in g/ml (in this case included in normahzed intensity), z1 the number of moles of electrons per gram of dissolved substance, and c2 the electron density of the solvent (Kratky, 1963). The quantity IO/P,, is the ratio between I,, the slit corrected, normalized intensity (I/c) at zero angle (extrapolated linearly to zero concentration) and P,, the intensity of the primary beam. The value of IO/P, was determined with the use of a Lupolen standard sample (Kratky et al., 1966). The Lupolen sample had been calibrated previously at the Graz Institut fi?r Physikahsche Chemie (Kratky & Wawra, 1963). The partial specific volume, v,, was determined by a precise digital densitometer developed by Kratky et al. (1969) (see also Stabinger et aZ., 196’7). For the coneentration range 5.2 to 147 mg enzyme/cm3 it was found that the value of fil was equal to 0732&0.013 cm3/g. Thus, the uncertainty in
TABLE

1

Molecular parameters of 1ysine:tmnsfer RNA ligate (yea&) Radius of gyration, R, Molecular weight, M by small-angle X-ray scattering? by equilibrium sedimentationf$ Vohme, Tr Degree of swelling, p

37.5 A 1-14x 105 1.38 x 106 2.95 x 105 Ae 2.12 (corresponds to 0.82 g of water per 1 g of protein)

t G1 = O-734cm3 g-l.

$ Rymo et al. (1972).

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ET

AL.

The volume, V, was determined to be 2.95x lo5 A3, using the following equation V = O-582 a31&.

(2)

Here 0.582 is a constant corresponding to the CuKa wavelength, h = 1.541 A, and Q is Porod’s invariant, which is defined by the equation & = j?rn 0

dm.

The quantities g and f are defined as Q and I, except that they have not been corrected for the collimation effect; m is the distance between the detector slit and the primary beam in the plane of registration (in cm). Q”was calculated from the integral over all the distances m for the product of the scattering intensity and the quantity, m. For m > O-6 cm the scattering curve is proportional to l/m3. The surface area of the particle was determined to be 3.05 x lo4 A, using the following equation (cf. Kratky, 1963): s=

Y x 87r x wa x lim (Im)” 112-s@=

Axax

’

(4)

where w, is the volume fraction of the solvent, and lim (r*m3) the limiting value obtained when I-m3 is plotted against m3. In this case, the plot approximately followed a straight line parallel to the m-axis, for m > 0.6 cm, and thus lim (I”.m3) m+@ is a constant. The degree of swelling, q, was calculated from the equation (Kratky, 1963) :

x v/M= 2 12 4=NA u1 x 1024 . ’ where N, stands for Avogadro’s constant. The value of q obtained corresponds to 0.82 g of water per 1 g of enzyme protein. This corresponds to a water content of 45.0% (w/w) for the protein molecule. (c) Shape of the enzyme

When the experimental scattering curve was compared with the theoretical curves calculated assuming one single type of triaxial body, only the curves calculated for ellipsoids were consistent with the experimentally obtained volume and radius of gyration. All calculations were made with an IBM 360165 computer, using a set of computer programs which were written according to the theory developed by Mittelbath & Porod (1961a,b,1962). The best agreement between the experimentally determined intensity curve and the theoretical curves was obtained for an ellipsoid model with axial ratios of 1.25: 1:0*47 (Fig. 2). For a radius of gyration of 37.5 A these ratios correspond to the semiaxes: A = 62.7 A, B = 50.1 A and C = 23.5 A. The volume of this body is 3.09 x lo5 A3 and its surface area is 2.6 x lo* AZ. It should be noted that our data do not indicate any major fluctuations in electron density within the enzyme molecule, such as would be expected for a large hole or a cleft, the size of a tRNA molecule. Thus, the intensity curve does not contain any detectable side maximum and it becomes proportional to 1/(29)a for 28 > 0.030 radians.

X-RAY

SCATTERING

OF LYXINE-tRNA

LIGASE

log hR Fm. 2. Slit corrected, normalized intensity curve (dottedline) extrapolated to zero concentration, compared with calculated intensity curves assuming ellipsoid models with the axial ratio 1.25 : I : 3/B. The values for the ratio of the C and B-axes, C/B, are as indicated. F, = (2n/h)2 sin 6, where X is the wavelength used, X = 1.54 A, and 0 is the half scattering angle. All the curves have been normalized to the same radius of gyration, R,.

On the other hand, a more detailed interpretation does not seem possible on the basis of the present data. This would have required measurements at high angles using high concentrations of enzyme to ensure sufficient accuracy. Unfortunatelyiy, she enzyme is not soluble at such concentrations (Rymo et al., 1970), and consequently we did not attempt a complete interpretation of data at angles larger than 40 milliradians. It is, however, interesting to note that the surface area of the enzyme is 16% larger than that of the ellipsoid model, indicating that the surface of the enzyme is not that of a smooth ellipsoid. A better agreement is obtained if w-e assume that each subunit of the molecule corresponds to one ellipsoid. We then obtain a surface area for two ellipsoids, which are close together within a volume of 295,000 A3, of 29,700 AZ, which is only 3% less than the experimental value. These data are consistent wit’h a molecule made up of two ellipsoid subunits with a groove between the subu.nits.

4. Discussion It is necessary to work with a solution of monodisperse particles in order to ensure an accurate interpretation of the X-ray small-angle scattering data (Gmnier Car Fournet, 1955). The highly purified enzyme used in this investigation was homogeneous as judged by polyacrylamide gel electrophoresis and ultracentrifugation (Rymo et al., 1972). 0ur results are consistent with previously reported parameters of this enzyme. Rymo et al. (1972) determined the molecular weight to be 138,000, based on equili-

158

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ET AL.

brium sedimentation and gel filtration studies. This value is larger than the present value of 114,000 (Table l), which may indicate that in solution the outer solvate envelope contributes about 17% to the total molecular weight of the enzyme. Crystals of the enzyme are trigonal and the parameters of the unit cell are a = 118 A and c = 190 A (Lagerkvist et al., 1972). The dimensions, 125 A x 100 A x 47 A, of our ellipsoid model are in agreement to within 6% with the cell parameters of the single crystal. We may tentatively suggest that the 125 A axis of the ellipsoid particle coincides with the a-axis of 118 A, and that the 100 A axis of the ellipsoid corresponds to half of the c-axis, 95 A. Chirikjian et al. (1972) report that the 1eucine:tRNA ligase is a dimer with a molecular weight of 120,000, a volume of 2.82 x lo5 A3, and a water content of 40.8%. Thus, the molecular parameters of the 1eucine:tRNA ligase are very similar to those of the 1ysine:tRNA ligase reported in this paper (Table 1). We thank Professor Dr 0. Kratky, Institut fiir Physikalische Chemie der Universitiit, Graz, for the Lupolen sample. This work was supported by a grant from the Swedish Natural Science Research Council. REFERENCES Chirikjian, J. G., Wright, H. T. & Fresco, J. R. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 1638. Guinier, A. & Fournet, G. (1955). Small-Angle Scattering of X-rays. John Wiley & Sons, Inc., New York. Heine, S. (1963). Acta Php. Austriaca, 16, 144. K&ten, W. J. (1971). Microchem. J. 16, 610. Kratky, 0. (1958). 2. Elektrochem. 62, 66. Kratky, 0. (1963). Progr. Biophys. 13, 105. Kratky, 0. & Skala, Z. (1958). 2. Elektrochem. 62, 73. Kratky, 0. & Wawra, H. (1963). Molzatsh. Chem. 94, 981. Kratky, O., Pilz, I. & Schmitz, P. J. (1966). J. Colloid. Sci. 21, 24. Kratky, O., Leopold, H. & Stabinger, H. (1969). 2. Angew. Phys. 27, 273. Lagerkvist, U., Rymo, L., Lindqvist, 0. & Andersson, E. (1972). J. Biol. Chem. 247, 3897. Leopold, H. (1968). 2. Angew. Phys. 25, 81. Mittelbach, P. & Porod, G. (1961a). Acta Phys. Austriaca, 14, 185. Mittelbach, P. & Porod, G. (1961b). Acta Phys. Austriaca, 14, 405. Mittelbach, P. & Porod, 0. (1962). Acta Phys. A?nstriaca, 15, 122. Rymo, L., Lagerkvist, U. & Wonacott, A. (1970). J. Biol. Chem. 245, 4308. Rymo, L., Lundvik, L. & Lagerkvist, U. (1972). J. Biol. Chem. 247, 3888. Stabinger, H., Leopold, H. & Kratky, 0. (1967). Monatsh. Chem. 98, 436. Strid, L. (1961). Actu Chem. Stand. 15, 1423. Waller, J.-P., Risler, J.-L., Monteilhet, C. & Zelwer, C. (1971). FEE% Letters, 16, 186.