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Molecular orientation and position of the pig M4 and H4 isoenzymes of lactate dehydrogenase in their crystal cells
J. Mol. Bid. (1973) 78, 665-673
Molecular Orientation and Position of the Pig m and H, Isoenzymes of Lactate Dehydrogenase in their Crystal Cells MARVIN L. HAUEEBT, GEOFFREYC. FORD AND MICHAEL G. ROSSMANN Department of BiologiudScierws,
Purdu~~University, West Lafayette, Ind. 47907, U.S.A.
(Received 30 March 1973, and in revised form 16 May 1973) Ternary complexes of M, and H4 &enzymes of porcine lactate dehydrogenase have been crystallized, the M4 isoenzyme in space group P22121 with one half molecule per asymmetric unit, and the H4 isoenzyme in space group C2 with one whole molecule per asymmetric unit. The orientation and position of the tetramers in their unit cells have been determined by X-ray analysis. Rotation function results oompariug the ternary complexes of the pig M4 isoenzyme with the known structure of the dog&h Bid enzyme not only defined the direction but also permitted recognition of the individual P, & and R molecular 2-fold axes. The position of the molecular center was determined by placing a properly oriented dog&h M, lactate dehydrogensse electron density into the pig muscle cell. Structure factors were calculated ss the molecular center wsa varied along the common crystallographic and molecular 2-fold axis and compared with observed amplitudes. Precession photographs of the three major zones of the monoclinic pig H, isoenzyme exhibited striking similarities to the corresponding zones of the orthorhombia pig Md isoenzyme, in spite of the differences in space groups. These similarities permit the determination of approximate phases from the implied orientation and position of the pig H, lactate dehydrogenase molecule in its monoclinic cell.
1. Introduction Lactate dehydrogenasse (EC 1.l .1.7) is a multisubunit NAD + -dependent enzyme that catalyses the interconversion of lactate and pyruvate in the glycolytic pathway. In most vertebrates, the enzyme is a tetramer of molecular weight 144,969. The tetramer contains four polypeptide chains with one active site per subunit (Heck, 1969 ; Schwert et al., 1967). Five isoenzymes with different electrophoretic mobilities result from the hybridization of the two parent subunit types abundant in most tissues (Wieland & Pfleiderer, 1957 ; Markert & Moller, 1959). The H subunit type is predominant in heart tissue and the M subunit type in skeletal muscle tissue. The enzymatic, immunological, physical and chemical properties are more alike among H, (LDH-1) or among M4 (LDH-5) isoensymes from different species than between the H, and M, forms of the same species (Markert & Apella, 1963; Pesce et al., 1967). These differences have been attributed to the different physiological roles of the two isoenzymes (Kaplan et al., 1968). The structure of the M, isoenzyme of lactate dehydrogenase from dogfish (Squ&s acanthizcs) has been determined by high resolution (Rossmann et al., 1971). The structure determination of an H, isoensyme was initiated to investigate the molecular basis of the functional differences of the LDHt isoenzymes. The suitabilities t Abbreviation
used: LDH,
la.otate dehydrogenam 666
666
M. L.
HACKERT,
G. C. FORD
AND
M. G. ROSSMANN
of a number of species were surveyed because dogfish hearts are too small to obtain easily a sufficient quantity of tissue. The pig isoenzymes were selected because suitable crystals of both were available, a large number of biochemical and sequence studies had been reported (Pfleiderer et al., 1970) and the source was readily obtainable. Once the structure of one member of a group of related proteins has been solved by conventional methods, the structures of other members are determined more readily. The known structure can be used as a model to calculate phases for the observed diffraction data from another member of the group. Electron density maps derived from such phases can be used to determine heavy atom derivative positions directly and also provide information rapidly about larger similarities and differences between the two related molecules.
2. Experimental Preliminary crystallization studies of pig LDH were carried out in Bristol by Drs R. A. Stinson and H. Gutfreund. They obtained some crystals large enough for preliminary diffraction studies of the isomorphous NAD-pyruvate and NADH/oxamate ternary complexes of pig M, LDH isoenzyme. Similar diamond-shaped plates with approximate dimensions of 1.0 mm x 0*5 mm x 0.16 mm have been grown in our laboratory at room temperature under the following conditions: LDH, 5 mg/ml; 0.0003 M-NADH; O-030 Moxamate and l-65 ~-ammonium sulfate, pH 8.0. Workable crystals of the pig H, LDH ternary complexes were obtained only after the acetone step common in earlier preparation procedures was replaced by successive chromatography on DEAE-cellulose and Sephadex ion-exchange columns. Isomorphous crystals of the ternary inhibitor complexes NADH/oxamatc and NAD/oxalate were obtained at room temperature under the following conditions: LDH, 6 mg/ml; 0.001 M-NADH or 0*004 M-NAD ; 0.030 r+oxamate or O-015 M-oxalate in 1.85 M-t&mmOniUm sulfate, pH 7.8. The morphology of the pig H4 LDH crystals is similar to that of the muscle crystals, except that 2 of the diamond points are truncated.
3. Results and Discussion (a) Relationship of pig M4 and pig H* lactate dehydrogenase cy&d.s Ternary inhibitor crystal forms :
complexes of the M, and H, LDH isoenzymes gave different
Pig M, LDH/NAD-pyruvate
P22,2,
Pig M4 LDH/NADH/oxamate Pig M, LDH/NAD/oxalate
b = 60.0 A 2=2
c = 136-2 A
I ~2
Pig H, LDH/NADH/oxamate Pig H, LDH/NAD/oxalate
a = 86-O A
I
a = 163.4 A b = 60.7 L% c = 138.6 A z=4 /9 = 93”
In addition to the lattice constants, a striking similarity exists when the diffraction patterns of the major zones of these two isoenzymes are compared, as shown in Plate I. This can be interpreted in terms of different packing of similarly oriented tetramers in the two systems. Consider a hypothetical C222, pig M4 LDH unit cell formed by doubling the pig M4 unit cell a-axis and introducing C centering (Fig. 1). Structure factor expressions given in Table 1 indicate that such a structure would produce intensity distributions similar to that observed for the pig H, isoenzyme,
Y
2 0
t
Y 2 0
b _-
c
0 f
c
e
ISOENZYMES
OF LACTATE
667
DEHYDROGENASE
Pig ht4 P22121 Z-2 1
1
FIQ. 1. Implied packing relationship of pig M, and H4 LDH molecules in their unit cells. A small reatangle represents one dimer with + and - indicating subunits. Diagonal lines indicate molecules translated by x/2 in unit cell. A large rectangle represents a tetramer. Axial directions are aa in Fig. 4.
TABLET Structure factor analysis indic&ng the agreement of the effect of the implied packing with the observed diffra&m results Assume: Pig H4 = pig M, + C centering a= = 2a*
Fhto = .Yf (janfm(rra~)+k(Yib))
h’+k=tn+l
4N12
h’=2h
(l+*anKlh’+ku2)) Fi%o = 2 f *anl(h’(zfa~)+R(Y/b)) PO% =
Fhc =
0
k = 2n+1
2F&
k = 2n
’ 53%
v=2?&+1 h’ = 2h
although such a hypothetical structure would still have a dimer as the asymmetric unit. The pig H, LDH unit cell can be approximated by doubling the pig M4 a-axis, introducing C centering, but with simultaneous loss of the crystallographic, molecular 2-fold axis. This reduces the space group symmetry from the hypothetical C222, to the observed C2 with a slight distortion of the p angle. There is now a tetramer, rather than a dime,, per asymmetric unit. Thus, knowledge of the orientation and position of the LDH molecule in the pig M, cell (where one of the molecular 2-fold axes is coincident with a crystallographic 2-fold axis) leads directly to the determination of the more difficult pig H, LDH structure. The rotation function (Rossmann BEBlow, 1962) was used to establish molecular symmetry axes and their orientation in the pig M, LDH crystallographic unit cell. Because of the 222 molecular symmetry found in dog&h M, LDH, the approximate 2-fold symmetry of the soluble malate dehydrogenase dimer (Tsernoglou et d., 1972), and the approximate 222 symmetry
668
M. L.
HACKERT,
G. C. FORD
AND
M.
G. ROSSMANN
found in glyceraldehyde-3-phosphate dehydrogenase (Rossmann et al., 1972; Gorjunov e.tal., 1972), it was assumed that only molecular 2-fold axes need be considered. Low resolution data (6.0 A) were collected by diffractometer methods on the native crystals of the pig M, LDH/NADH/ oxamate ternary complex. The rotation function was used to compare the pig M, LDH Patterson with itself to locate the directions of the other two molecular 2-fold axes. Only the data from 15 to 6 A resolution were used in the comparison. The original Patterson contained 1259 terms modified to remove the origin peak. It was compared to a large term Patterson of 331 unmodified terms. A radius of integration of only 54 A had to be used for the comparison in order to avoid overlap of neighboring origins. In all calculations, 27 nearest neighbors of each non-integral reciprocal lattice point were used for interpolation. A check run, using the nearest 125 neighboring reciprocal lattice points, did not change the relative positions of peaks in the rotation function. Because the space group symmetry fixes the position of one molecular 2-fold axis, the other two molecular d-fold axes must lie in the plane normal to this crystallographic diad axis. This reduces the necessary search to a line defined in polar co-ordinates by $ = 270”, K = 180” and 0 5 I$ 2 45”. The results in Figure 2 (# was stepped in 2.5” intervals) show only one large peak (at 4 = 13”) other than the origin. Absence of a very large peak (about half origin height) in the Harker sections ruled out the possibility of the molecular noncrystallographic axes running parallel to the crystallographic axes and, hence, their direction being hidden in the origin peak of the rotation function. The equivalent
FIG. 2. (a) Rotation function of pig M, LDH for + = 270”. Resolution of data radius of integration = 64 A. The 3 curves correspond to K = 180’ (-O-O-), and QO’(-A-A-). (b) Comparison rotation fun&ion of pig M, LDH with dogfkh 3 owes correspond to superimposing the pig M, orystallogmpbio 2-fold axis on moleoular I’(-()-@), R(-A-A-) and Q(-O-O-) axes.
ws8 6.0 A with 120’ (-u-Cl-) Mb LDH. The the dog%h Md
ISOENZYMES
OF LACTATE
DEHYDROGENASE
669
searches for 4-fold and 3-fold axes are shown merely to indicate background readings. To verify that the non-origin peak in the rotation function was truly a single peak and not a symmetry build up of smaller peaks just off the 4 = 270” plane, $ and $I were stepped in 10” intervals to give a coarse exploration of the K = 180” section of the rotation function (Fig. 3). Inspection of the Ok1diffraction patterns reveals an intense region near 063 and 064, which forms a molecular axis spike at 13” to the crystallographic b-axis, in agreement with the rotation results. 6
FIa. 3. Stereographic projection of the rotation coarse search in I) and r$ at 10” intervals.
function
of pig M., LDH
for K = 180” with
The three molecular 2-fold axes in dogfish M, LDH have been labeled P, Q and R (Rossmann et al., 1973). Evidence for the conservation of the tertiary and quaternary structure among the dehydrogenases was found in the structure of soluble malate dehydrogenase. Although only a dimer, it possessesa non-crystallographic S-fold axis nearly identical to the Q molecular 2-fold axis of dogfish M, LDH. Assuming that the dogfish and pig M, LDH ternary complexes would be very similar, they were compared not only to re-affirm the positioning of the molecular 2-fold axis, but also to identify the individual P, Q and R molecular 2-fold axes, The known electron density of one tetramer of the abortive ternary complex of dogfish LDH was cut out and placed in an oversized unit cell of a = b = c = 160 A in space group P222. The molecular center was placed at (O,O,O)with its molecular P, Q and R axes coincident with the a, - c, b axes, respectively. Structure factors were computed to 6 A resolution from the map based on 5 A phases and sampled at approximately 2 A intervals. As the largest molecular diameter is approximately 80 A, the oversized cell prevented overlap of the self-Patterson at neighboring origins. The conditions for the pig M, LDH-dogfish M, LDH rotation function were similar to those used previously. The Patterson of the P222 cell was represented by the 363 large terms greater than 2.0 times the mean intensity in each of six ranges of sin 8. The dogfish M, LDH P, Q and R 2-fold axes were then aligned in turn with the one crystallographic 2-fold axis in the pig muscle LDH cell. The line of the rotation function to be searched in each instance is 0 2 K 5 90”, 4 = 90” and + = 0” (Fig. 2(b)). 44
670
M. L.
HACKERT,
G. C. FORD
AND
M. ,G. ROSSMANN
The peak at K = 13”, when the dogfish M, LDH P axis was aligned with the crystallographic 2-fold axis of pig M, LDH, is more than twice the height of any other peak. The angle of 13” agrees with the previously determined orientation (without identification) based only on pig muscle LDH data. This alignment corresponds to rotating the R and Q molecular axes 13” from the b and -c axes, respectively, in the pig muscle cell. Using an ORTEP drawing (Johnson, 1965) of the dogfish LDH tetramer projected along the P molecular axis, the implied packing in the pig muscle cell is shown in Figure 4.
k Fm. 4. ORTEP LDH unit cell.
drawing
of the z projection
55 - r;, p, :: a : I : : ’ 45 -
of the implied
packing
of tetramers
in the pig M,
.“‘-- ----._ ,,__,-_.,,-1 :2% ,, I :,,+; : : ’ /
k
35 I
Jr-----
, 0
I 01
I
I
02
03
I
04
I
0.5
FIG. 6. Transletion search along the crystallographic 2-fold axis for the position of the molecular center: spherical molecular envelope (- - - - - -) and true molecular boundary from den). Resolution of the data was 8.0 A. sity cut-out (R
=
(IF01
2PQl
IPel)
ISOENZYMES
OF LACTATE
DEHYDROGENASE
671
FIQ. 6. Comparison of the 6.0 A resolution eleotron density of the 2 independent active sites in the pig M, LDH/NADH/oxamate complex. The backbone co-ordinates taken are from the corresponding dogfish &ix, LDH ternary struoture. The electron density is bssed on phaeee odoulated on the basis of the known dogfish M, LDH structure and on the observed pig M4 LDH amplitudes.
ti72
M. I,.
HACKERT,
G. C. 1pORIl
ANI,
M. C:. KOSSMANN
(b) Po&ion of the molecular center along the 2-fold a&s A properly oriented cut-out of the dogfish M, LDH ternary complex density can be placed in the pig M, cell and the corresponding structure factors calculated. Assuming a similarity between these two structures, the best agreement between the observed and calculated amplitudes should occur when the molecular center of the dogfish LDH molecule has been correctly positioned in the pig muscle cell. This method is similar to that reported previously by Tollin (1966,1969), Joynson et al. (1970), Rossmann et al. (1972) and Crowhher & Blow (1967). In this case, however, a conventional agreement factor was evaluated for each trial position of the molecular center, since the search could be restricted between the limits 0 F: x < 0.5 along the S-fold axis. Data from 20 to 8 A resolution were used in the calculation. The calculated amplitudes were obtained by two procedures, each scaled to the observed terms by equating the sums of their respective amplitudes. The calculated structure factor amplitudes were first obtained by interpolating the previous set in reciprocal sfJace, assuming a sphere of radius 30 A as the molecular envelope. An alternative structure factor calculation was done by placing the cut-out of the properly oriented dogfish molecular density in the pig muscle cell ,and doing the Fourier transform. The agreement of both these methods is indicated in Figure 5. The sharp minimum at x = 0.078 has a half-width of about 2.5 A, indicating the high precision for the correct positioning of the molecular center. An electron density map for pig M, LDH/NADH/oxamate was computed using the observed amplitudes with calculated phases based on the dogfish M, LDH/NADpyruvate electron density map. A section by section comparison of the new density map with a superimposed dogfish LDH ternary co-ordinate backbone was made to check for differences between the pig and dogfish muscle complexes, and between the two independent subunits in the pig muscle complex. A subjective comparison (at this resolution) did not show any significant differences. The low B-factor of 36% is itself indicative of the similarity of these two structures. The two independent active site regions from this map are shown in Figure 6. The loop is found in a down or closed configuration at each site, although the contouring schemes used give the impression of more loop density in one case than the other. Only at higher resolution, where the individual differences in amino acid sequence between dogfish and pig muscle LDH should be recognizable, will there be any discernible differences in these structures.
4. Conclusion The orientation and position of the pig M, LDH molecule in its P22,2, cell have been determined. From these results, trial pig M, LDH structure factor phases have been calculated, based on the proper positioning of the dogfish LDH density in the pig muscle cell. Extension of these results through packing relationships also resulted in trial phases for the pig H, LDH structure, where a tetramer of molecular weight 144,000 composes the asymmetric unit. Such phases should not only lead to the direct determination of heavy atom derivative positions, but also could be applied to high resolution native data when initially comparing these related structures for their gross differences. We thank Dr M. J. Adams for her advice and assistance during the early stages of this work and Mm Sharon Wilder for technical assistance in prepariug this manuscript. This
ISOENZYMES
OF LACTATE
DEHYDROGENASE
673
work was supported by National Science Foundation grant no. GB29696x and National Institutes of Health grant no. GM10704. One of us (M. L. H.) was the recipient of a fellowship from the National Institutes of Health (no. 1 F02 AM 62315-01). REFERENCES Crowther, R. A. & Blow, D. M. (1967). Acta @/8tdOg. 23, 544648. Gorjunov, A. I., Andreeva, N. S., Baranowski, T. & Wolny, M. (1972). J. Mol. Biol. 69, 421-426. Heck, H. d’A. (1969). J. Biol. Chem. 244, 437g-4381. Johnson, C. K. (1966). Document ORNL-3794, Revised, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A. doynson, M. A., North, A. C. T., Sarma, V. R., Dickerson, R. E. & Steinrauf, L. K. (1970). J. Mol. Biol. 50, 137-142. Kaplan, N. O., Everse, J. & Admiraal, J. (1968). Ann. N.Y. Ad. Sci. 151, 400-412. Marker& C. L. & Appella, E. (1963). Ann. N.Y. Au-d. Sci. 103, 915-929. Marker& C. L. & Moller, F. (1969). Proc. Nat. AC&. Sci., U.S.A. 45, 763-763. Pesce, A. J., Fondy, T. P., Stolzenbach, F. G., Castillo, F. & Kaplan, N. 0. (1967). J. Biol. Chena. 242, 2151-2167. Pfleiderer, G., Woenckhaus, C. J., Jeckel, D. & Mella, K. (1970). In Pyridine Nudeotidedependent Dehydrogenmes (Sund, H., ed.), pp. 145-155. Springer-Verlag, Heidelberg, West Germany. Rossmann, M. G. & Blow, D. M. (1962). Acta Gry&uZZog. 15, 24-31. Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Lentz, P. J., Jr, McPherson, A., Jr, Schevitz, R. W. & Smiley, I. E. (1971). Cold Spr&g Harbor Symp. Qwmt. Biol. 36, 179-191. Rossmann, M. G., Ford, G. C., Watson, H. C. & Banaszak, L. J. (1972). J. Mol. BioE. 64, 237-249.
Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Liljas, A., Rao, S. T., Banaszak, L. J., Hill, E., Tsernoglou, D. & Webb, L. (1973). J. Mol. Biol.
76, 533-537.
Schwert, G. W., Miller, B. R. & Peanasky, R. J. (1967). J. Bid. Chern. 242, 3245-3252. Tollin, P. (1966). Aota ‘7?yetdOg. 21, 613-614. Tollin, P. (1969). J. Mol. Bid. 45, 481-490. Tsernoglou, D., Hill, E. & Banaszak, L. J. (1972). J. Mol. Bid 69, 75-87. Wieland, Th. t Pfleiderer, G. (1967). Biochem. 2. 329, 112-116.
Molecular Orientation and Position of the Pig m and H, Isoenzymes of Lactate Dehydrogenase in their Crystal Cells MARVIN L. HAUEEBT, GEOFFREYC. FORD AND MICHAEL G. ROSSMANN Department of BiologiudScierws,
Purdu~~University, West Lafayette, Ind. 47907, U.S.A.
(Received 30 March 1973, and in revised form 16 May 1973) Ternary complexes of M, and H4 &enzymes of porcine lactate dehydrogenase have been crystallized, the M4 isoenzyme in space group P22121 with one half molecule per asymmetric unit, and the H4 isoenzyme in space group C2 with one whole molecule per asymmetric unit. The orientation and position of the tetramers in their unit cells have been determined by X-ray analysis. Rotation function results oompariug the ternary complexes of the pig M4 isoenzyme with the known structure of the dog&h Bid enzyme not only defined the direction but also permitted recognition of the individual P, & and R molecular 2-fold axes. The position of the molecular center was determined by placing a properly oriented dog&h M, lactate dehydrogensse electron density into the pig muscle cell. Structure factors were calculated ss the molecular center wsa varied along the common crystallographic and molecular 2-fold axis and compared with observed amplitudes. Precession photographs of the three major zones of the monoclinic pig H, isoenzyme exhibited striking similarities to the corresponding zones of the orthorhombia pig Md isoenzyme, in spite of the differences in space groups. These similarities permit the determination of approximate phases from the implied orientation and position of the pig H, lactate dehydrogenase molecule in its monoclinic cell.
1. Introduction Lactate dehydrogenasse (EC 1.l .1.7) is a multisubunit NAD + -dependent enzyme that catalyses the interconversion of lactate and pyruvate in the glycolytic pathway. In most vertebrates, the enzyme is a tetramer of molecular weight 144,969. The tetramer contains four polypeptide chains with one active site per subunit (Heck, 1969 ; Schwert et al., 1967). Five isoenzymes with different electrophoretic mobilities result from the hybridization of the two parent subunit types abundant in most tissues (Wieland & Pfleiderer, 1957 ; Markert & Moller, 1959). The H subunit type is predominant in heart tissue and the M subunit type in skeletal muscle tissue. The enzymatic, immunological, physical and chemical properties are more alike among H, (LDH-1) or among M4 (LDH-5) isoensymes from different species than between the H, and M, forms of the same species (Markert & Apella, 1963; Pesce et al., 1967). These differences have been attributed to the different physiological roles of the two isoenzymes (Kaplan et al., 1968). The structure of the M, isoenzyme of lactate dehydrogenase from dogfish (Squ&s acanthizcs) has been determined by high resolution (Rossmann et al., 1971). The structure determination of an H, isoensyme was initiated to investigate the molecular basis of the functional differences of the LDHt isoenzymes. The suitabilities t Abbreviation
used: LDH,
la.otate dehydrogenam 666
666
M. L.
HACKERT,
G. C. FORD
AND
M. G. ROSSMANN
of a number of species were surveyed because dogfish hearts are too small to obtain easily a sufficient quantity of tissue. The pig isoenzymes were selected because suitable crystals of both were available, a large number of biochemical and sequence studies had been reported (Pfleiderer et al., 1970) and the source was readily obtainable. Once the structure of one member of a group of related proteins has been solved by conventional methods, the structures of other members are determined more readily. The known structure can be used as a model to calculate phases for the observed diffraction data from another member of the group. Electron density maps derived from such phases can be used to determine heavy atom derivative positions directly and also provide information rapidly about larger similarities and differences between the two related molecules.
2. Experimental Preliminary crystallization studies of pig LDH were carried out in Bristol by Drs R. A. Stinson and H. Gutfreund. They obtained some crystals large enough for preliminary diffraction studies of the isomorphous NAD-pyruvate and NADH/oxamate ternary complexes of pig M, LDH isoenzyme. Similar diamond-shaped plates with approximate dimensions of 1.0 mm x 0*5 mm x 0.16 mm have been grown in our laboratory at room temperature under the following conditions: LDH, 5 mg/ml; 0.0003 M-NADH; O-030 Moxamate and l-65 ~-ammonium sulfate, pH 8.0. Workable crystals of the pig H, LDH ternary complexes were obtained only after the acetone step common in earlier preparation procedures was replaced by successive chromatography on DEAE-cellulose and Sephadex ion-exchange columns. Isomorphous crystals of the ternary inhibitor complexes NADH/oxamatc and NAD/oxalate were obtained at room temperature under the following conditions: LDH, 6 mg/ml; 0.001 M-NADH or 0*004 M-NAD ; 0.030 r+oxamate or O-015 M-oxalate in 1.85 M-t&mmOniUm sulfate, pH 7.8. The morphology of the pig H4 LDH crystals is similar to that of the muscle crystals, except that 2 of the diamond points are truncated.
3. Results and Discussion (a) Relationship of pig M4 and pig H* lactate dehydrogenase cy&d.s Ternary inhibitor crystal forms :
complexes of the M, and H, LDH isoenzymes gave different
Pig M, LDH/NAD-pyruvate
P22,2,
Pig M4 LDH/NADH/oxamate Pig M, LDH/NAD/oxalate
b = 60.0 A 2=2
c = 136-2 A
I ~2
Pig H, LDH/NADH/oxamate Pig H, LDH/NAD/oxalate
a = 86-O A
I
a = 163.4 A b = 60.7 L% c = 138.6 A z=4 /9 = 93”
In addition to the lattice constants, a striking similarity exists when the diffraction patterns of the major zones of these two isoenzymes are compared, as shown in Plate I. This can be interpreted in terms of different packing of similarly oriented tetramers in the two systems. Consider a hypothetical C222, pig M4 LDH unit cell formed by doubling the pig M4 unit cell a-axis and introducing C centering (Fig. 1). Structure factor expressions given in Table 1 indicate that such a structure would produce intensity distributions similar to that observed for the pig H, isoenzyme,
Y
2 0
t
Y 2 0
b _-
c
0 f
c
e
ISOENZYMES
OF LACTATE
667
DEHYDROGENASE
Pig ht4 P22121 Z-2 1
1
FIQ. 1. Implied packing relationship of pig M, and H4 LDH molecules in their unit cells. A small reatangle represents one dimer with + and - indicating subunits. Diagonal lines indicate molecules translated by x/2 in unit cell. A large rectangle represents a tetramer. Axial directions are aa in Fig. 4.
TABLET Structure factor analysis indic&ng the agreement of the effect of the implied packing with the observed diffra&m results Assume: Pig H4 = pig M, + C centering a= = 2a*
Fhto = .Yf (janfm(rra~)+k(Yib))
h’+k=tn+l
4N12
h’=2h
(l+*anKlh’+ku2)) Fi%o = 2 f *anl(h’(zfa~)+R(Y/b)) PO% =
Fhc =
0
k = 2n+1
2F&
k = 2n
’ 53%
v=2?&+1 h’ = 2h
although such a hypothetical structure would still have a dimer as the asymmetric unit. The pig H, LDH unit cell can be approximated by doubling the pig M4 a-axis, introducing C centering, but with simultaneous loss of the crystallographic, molecular 2-fold axis. This reduces the space group symmetry from the hypothetical C222, to the observed C2 with a slight distortion of the p angle. There is now a tetramer, rather than a dime,, per asymmetric unit. Thus, knowledge of the orientation and position of the LDH molecule in the pig M, cell (where one of the molecular 2-fold axes is coincident with a crystallographic 2-fold axis) leads directly to the determination of the more difficult pig H, LDH structure. The rotation function (Rossmann BEBlow, 1962) was used to establish molecular symmetry axes and their orientation in the pig M, LDH crystallographic unit cell. Because of the 222 molecular symmetry found in dog&h M, LDH, the approximate 2-fold symmetry of the soluble malate dehydrogenase dimer (Tsernoglou et d., 1972), and the approximate 222 symmetry
668
M. L.
HACKERT,
G. C. FORD
AND
M.
G. ROSSMANN
found in glyceraldehyde-3-phosphate dehydrogenase (Rossmann et al., 1972; Gorjunov e.tal., 1972), it was assumed that only molecular 2-fold axes need be considered. Low resolution data (6.0 A) were collected by diffractometer methods on the native crystals of the pig M, LDH/NADH/ oxamate ternary complex. The rotation function was used to compare the pig M, LDH Patterson with itself to locate the directions of the other two molecular 2-fold axes. Only the data from 15 to 6 A resolution were used in the comparison. The original Patterson contained 1259 terms modified to remove the origin peak. It was compared to a large term Patterson of 331 unmodified terms. A radius of integration of only 54 A had to be used for the comparison in order to avoid overlap of neighboring origins. In all calculations, 27 nearest neighbors of each non-integral reciprocal lattice point were used for interpolation. A check run, using the nearest 125 neighboring reciprocal lattice points, did not change the relative positions of peaks in the rotation function. Because the space group symmetry fixes the position of one molecular 2-fold axis, the other two molecular d-fold axes must lie in the plane normal to this crystallographic diad axis. This reduces the necessary search to a line defined in polar co-ordinates by $ = 270”, K = 180” and 0 5 I$ 2 45”. The results in Figure 2 (# was stepped in 2.5” intervals) show only one large peak (at 4 = 13”) other than the origin. Absence of a very large peak (about half origin height) in the Harker sections ruled out the possibility of the molecular noncrystallographic axes running parallel to the crystallographic axes and, hence, their direction being hidden in the origin peak of the rotation function. The equivalent
FIG. 2. (a) Rotation function of pig M, LDH for + = 270”. Resolution of data radius of integration = 64 A. The 3 curves correspond to K = 180’ (-O-O-), and QO’(-A-A-). (b) Comparison rotation fun&ion of pig M, LDH with dogfkh 3 owes correspond to superimposing the pig M, orystallogmpbio 2-fold axis on moleoular I’(-()-@), R(-A-A-) and Q(-O-O-) axes.
ws8 6.0 A with 120’ (-u-Cl-) Mb LDH. The the dog%h Md
ISOENZYMES
OF LACTATE
DEHYDROGENASE
669
searches for 4-fold and 3-fold axes are shown merely to indicate background readings. To verify that the non-origin peak in the rotation function was truly a single peak and not a symmetry build up of smaller peaks just off the 4 = 270” plane, $ and $I were stepped in 10” intervals to give a coarse exploration of the K = 180” section of the rotation function (Fig. 3). Inspection of the Ok1diffraction patterns reveals an intense region near 063 and 064, which forms a molecular axis spike at 13” to the crystallographic b-axis, in agreement with the rotation results. 6
FIa. 3. Stereographic projection of the rotation coarse search in I) and r$ at 10” intervals.
function
of pig M., LDH
for K = 180” with
The three molecular 2-fold axes in dogfish M, LDH have been labeled P, Q and R (Rossmann et al., 1973). Evidence for the conservation of the tertiary and quaternary structure among the dehydrogenases was found in the structure of soluble malate dehydrogenase. Although only a dimer, it possessesa non-crystallographic S-fold axis nearly identical to the Q molecular 2-fold axis of dogfish M, LDH. Assuming that the dogfish and pig M, LDH ternary complexes would be very similar, they were compared not only to re-affirm the positioning of the molecular 2-fold axis, but also to identify the individual P, Q and R molecular 2-fold axes, The known electron density of one tetramer of the abortive ternary complex of dogfish LDH was cut out and placed in an oversized unit cell of a = b = c = 160 A in space group P222. The molecular center was placed at (O,O,O)with its molecular P, Q and R axes coincident with the a, - c, b axes, respectively. Structure factors were computed to 6 A resolution from the map based on 5 A phases and sampled at approximately 2 A intervals. As the largest molecular diameter is approximately 80 A, the oversized cell prevented overlap of the self-Patterson at neighboring origins. The conditions for the pig M, LDH-dogfish M, LDH rotation function were similar to those used previously. The Patterson of the P222 cell was represented by the 363 large terms greater than 2.0 times the mean intensity in each of six ranges of sin 8. The dogfish M, LDH P, Q and R 2-fold axes were then aligned in turn with the one crystallographic 2-fold axis in the pig muscle LDH cell. The line of the rotation function to be searched in each instance is 0 2 K 5 90”, 4 = 90” and + = 0” (Fig. 2(b)). 44
670
M. L.
HACKERT,
G. C. FORD
AND
M. ,G. ROSSMANN
The peak at K = 13”, when the dogfish M, LDH P axis was aligned with the crystallographic 2-fold axis of pig M, LDH, is more than twice the height of any other peak. The angle of 13” agrees with the previously determined orientation (without identification) based only on pig muscle LDH data. This alignment corresponds to rotating the R and Q molecular axes 13” from the b and -c axes, respectively, in the pig muscle cell. Using an ORTEP drawing (Johnson, 1965) of the dogfish LDH tetramer projected along the P molecular axis, the implied packing in the pig muscle cell is shown in Figure 4.
k Fm. 4. ORTEP LDH unit cell.
drawing
of the z projection
55 - r;, p, :: a : I : : ’ 45 -
of the implied
packing
of tetramers
in the pig M,
.“‘-- ----._ ,,__,-_.,,-1 :2% ,, I :,,+; : : ’ /
k
35 I
Jr-----
, 0
I 01
I
I
02
03
I
04
I
0.5
FIG. 6. Transletion search along the crystallographic 2-fold axis for the position of the molecular center: spherical molecular envelope (- - - - - -) and true molecular boundary from den). Resolution of the data was 8.0 A. sity cut-out (R
=
(IF01
2PQl
IPel)
ISOENZYMES
OF LACTATE
DEHYDROGENASE
671
FIQ. 6. Comparison of the 6.0 A resolution eleotron density of the 2 independent active sites in the pig M, LDH/NADH/oxamate complex. The backbone co-ordinates taken are from the corresponding dogfish &ix, LDH ternary struoture. The electron density is bssed on phaeee odoulated on the basis of the known dogfish M, LDH structure and on the observed pig M4 LDH amplitudes.
ti72
M. I,.
HACKERT,
G. C. 1pORIl
ANI,
M. C:. KOSSMANN
(b) Po&ion of the molecular center along the 2-fold a&s A properly oriented cut-out of the dogfish M, LDH ternary complex density can be placed in the pig M, cell and the corresponding structure factors calculated. Assuming a similarity between these two structures, the best agreement between the observed and calculated amplitudes should occur when the molecular center of the dogfish LDH molecule has been correctly positioned in the pig muscle cell. This method is similar to that reported previously by Tollin (1966,1969), Joynson et al. (1970), Rossmann et al. (1972) and Crowhher & Blow (1967). In this case, however, a conventional agreement factor was evaluated for each trial position of the molecular center, since the search could be restricted between the limits 0 F: x < 0.5 along the S-fold axis. Data from 20 to 8 A resolution were used in the calculation. The calculated amplitudes were obtained by two procedures, each scaled to the observed terms by equating the sums of their respective amplitudes. The calculated structure factor amplitudes were first obtained by interpolating the previous set in reciprocal sfJace, assuming a sphere of radius 30 A as the molecular envelope. An alternative structure factor calculation was done by placing the cut-out of the properly oriented dogfish molecular density in the pig muscle cell ,and doing the Fourier transform. The agreement of both these methods is indicated in Figure 5. The sharp minimum at x = 0.078 has a half-width of about 2.5 A, indicating the high precision for the correct positioning of the molecular center. An electron density map for pig M, LDH/NADH/oxamate was computed using the observed amplitudes with calculated phases based on the dogfish M, LDH/NADpyruvate electron density map. A section by section comparison of the new density map with a superimposed dogfish LDH ternary co-ordinate backbone was made to check for differences between the pig and dogfish muscle complexes, and between the two independent subunits in the pig muscle complex. A subjective comparison (at this resolution) did not show any significant differences. The low B-factor of 36% is itself indicative of the similarity of these two structures. The two independent active site regions from this map are shown in Figure 6. The loop is found in a down or closed configuration at each site, although the contouring schemes used give the impression of more loop density in one case than the other. Only at higher resolution, where the individual differences in amino acid sequence between dogfish and pig muscle LDH should be recognizable, will there be any discernible differences in these structures.
4. Conclusion The orientation and position of the pig M, LDH molecule in its P22,2, cell have been determined. From these results, trial pig M, LDH structure factor phases have been calculated, based on the proper positioning of the dogfish LDH density in the pig muscle cell. Extension of these results through packing relationships also resulted in trial phases for the pig H, LDH structure, where a tetramer of molecular weight 144,000 composes the asymmetric unit. Such phases should not only lead to the direct determination of heavy atom derivative positions, but also could be applied to high resolution native data when initially comparing these related structures for their gross differences. We thank Dr M. J. Adams for her advice and assistance during the early stages of this work and Mm Sharon Wilder for technical assistance in prepariug this manuscript. This
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work was supported by National Science Foundation grant no. GB29696x and National Institutes of Health grant no. GM10704. One of us (M. L. H.) was the recipient of a fellowship from the National Institutes of Health (no. 1 F02 AM 62315-01). REFERENCES Crowther, R. A. & Blow, D. M. (1967). Acta @/8tdOg. 23, 544648. Gorjunov, A. I., Andreeva, N. S., Baranowski, T. & Wolny, M. (1972). J. Mol. Biol. 69, 421-426. Heck, H. d’A. (1969). J. Biol. Chem. 244, 437g-4381. Johnson, C. K. (1966). Document ORNL-3794, Revised, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A. doynson, M. A., North, A. C. T., Sarma, V. R., Dickerson, R. E. & Steinrauf, L. K. (1970). J. Mol. Biol. 50, 137-142. Kaplan, N. O., Everse, J. & Admiraal, J. (1968). Ann. N.Y. Ad. Sci. 151, 400-412. Marker& C. L. & Appella, E. (1963). Ann. N.Y. Au-d. Sci. 103, 915-929. Marker& C. L. & Moller, F. (1969). Proc. Nat. AC&. Sci., U.S.A. 45, 763-763. Pesce, A. J., Fondy, T. P., Stolzenbach, F. G., Castillo, F. & Kaplan, N. 0. (1967). J. Biol. Chena. 242, 2151-2167. Pfleiderer, G., Woenckhaus, C. J., Jeckel, D. & Mella, K. (1970). In Pyridine Nudeotidedependent Dehydrogenmes (Sund, H., ed.), pp. 145-155. Springer-Verlag, Heidelberg, West Germany. Rossmann, M. G. & Blow, D. M. (1962). Acta Gry&uZZog. 15, 24-31. Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Lentz, P. J., Jr, McPherson, A., Jr, Schevitz, R. W. & Smiley, I. E. (1971). Cold Spr&g Harbor Symp. Qwmt. Biol. 36, 179-191. Rossmann, M. G., Ford, G. C., Watson, H. C. & Banaszak, L. J. (1972). J. Mol. BioE. 64, 237-249.
Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hackert, M. L., Liljas, A., Rao, S. T., Banaszak, L. J., Hill, E., Tsernoglou, D. & Webb, L. (1973). J. Mol. Biol.
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Schwert, G. W., Miller, B. R. & Peanasky, R. J. (1967). J. Bid. Chern. 242, 3245-3252. Tollin, P. (1966). Aota ‘7?yetdOg. 21, 613-614. Tollin, P. (1969). J. Mol. Bid. 45, 481-490. Tsernoglou, D., Hill, E. & Banaszak, L. J. (1972). J. Mol. Bid 69, 75-87. Wieland, Th. t Pfleiderer, G. (1967). Biochem. 2. 329, 112-116.