Leucine aminopeptidase (bovine lens): an electron microscopic study

JOURNAL OF ULTRASTRUCTURE RESEARCH

68,

9 2 - 1 0 0 (1979)

Leucine Aminopeptidase (Bovine Lens): An Electron Microscopic Study A L L E N TAYLOR, ] FREDERICK

H. CARPENTER, AND

ALEXANDER WLODAWER 2'*

Department of Biochemistry, University of California, Berkeley, California 94720, and* Department of Chemistry, Stanford University, Stanford, California 94305 Received October 23, 1978 The quaternary structure of leucine aminopeptidase was studied by electron microscopy of negatively stained single molecules and of both stained and unstained thin sections of crystals. The predominant images observed in micrographs of single molecules were triangles, many of which had a more dense triangle inset into the outer triangle and rotated by 60 ° from it. Measurements performed on electron micrographs of thin sections of the crystals cut parallel to the (0001) and (1120) planes gave unit cell parameters of a = 120/~ (+10 A) and c = 110/~ (±15 A). A quaternary structure composed of six asymmetric bilobal subunits arranged so that the principal lobes are eclipsed and the minor lobes are staggered in a manner similar to that of aspartate carbamoyltransferase is the model that best explains the images observed.

and Vahl, 1973; Thompson and Carpenter, 1976a,b; Kiselev et al., 1977). Several molecular models for bovine lens LAP have been proposed. Based on electron microscopic visualizations Kretschmer (1968) proposed that the molecule is a decamer comprised of two planar pentameric rings which form a cylinder with an axial ratio of 0.69. Low-angle X-ray diffraction studies (Kretschmer and Kollin, 1969) indicated that the cylinder was 66.7 /~ in height and that it had an outer diameter of 107.2/~ corresponding to an axial ratio of 0.62. Earlier hydrodynamic measurements, based on half-maximal hydration (Kretschmet and Hanson, 1965), indicated an axial ratio of 0.365 (oblate) or 2.65 (prolate). The form factor (f) determined by the solution X-ray studies (Kretschmer and Kollin, 1969) was 1.24 which is in good aggrement with the frictional coefficient fifo = 1.23 determined by sedimentation diffusion (Kretschmer and Hanson, 1965). The inside diameter was said to be 32.7/~ which would permit free passage of water. Crosslinking studies by Carpenter and Harrington (1972) indicated that the enzyme is a hexamer composed of a trimer of dimers and precluded a planar hexagonal arrangement of the subunits with all het-

The N-terminal exopeptidase leucine aminopeptidase (EC 3.4.11.1) was first observed in extracts of swine intestinal mucosa by LinderstrSm-Lang (1929). Its activity has been associated with cataract formation (Devi, 1963), tumor growth, hepatic diseases, and placental function (Uete et al., 1974). Other aminopeptidases of plant and animal origin have been described (Delange and Smith, 1971; Riordan, 1974); however, structural information about this group of proteases is scanty. The molecular weight of bovine lens leucine aminopeptidase has been determined to be about 326 000 by several groups (Kretschmer and Hanson, 1965; Melbye and Carpenter, 1971; Carpenter and Harrington, 1972). The number of subunits in leucine aminopeptidase (LAP) molecules has been a subject of some controversy. While 10 subunits have been indicated in several reports (Kretschmer, 1967a,b, 1968; Kretschmer and Hanson, 1968), the currently accepted number is 6 (Weber and Osborn, 1969; Melbye and Carpenter, 1971; Carpenter and Harrington, 1972; Carpenter 1Present address: Chemistry Department, Williams College, Williamston, Mass. 01267. 2 Present address: Reaction Radiation Division, National Bureau of Standards, Washington, D. C. 92 0022-5320/79/070092-09502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

ELECTRON MICROSCOPY OF LEUCINE AMINOPEPTIDASE

erologous interactions. Low-angle X-ray scattering studies of LAP in solution gave a radius of gyration of 44.5 A, the greatest diameter of the molecule of 125 A, and a subunit diameter of 54 /~ (Damaschun et al., 1973). This was reported to support a hexameric enzyme with an octahedral arrangement of spherical subunits. Subsequently, LAP images were interpreted as resulting from two partially staggered trimers (Wangermann et al., 1976) and finally, the foregoing image was said to be circumscribed by a less dense hexagonal image (Kiselev et al., 1977). Although they differ on the shape and size of the protomer, these latter two studies agree with the hexameric description of the molecule previously proposed by Carpenter and Harrington (1972) and seem to contradict the decameric model mentioned by Kretschmer (1968). Because of the contradictory reports on the number of subunits and the quaternary structure of the enzyme and because we were able to obtain suitably sized crystals, we endeavored to study the enzyme in its crystalline and solution forms. Preliminary results from X-ray diffraction studies have been published elsewhere (Jurnak et al., 1977). The present study presents electron microscopic images of single molecules and of thin sections of the crystalline enzyme cut parallel to the (0001) and (1130) planes. Based on these images and the preliminary X-ray data we have arrived at a model for the protomer and the arrangement of protomers in single molecules. MATERIALS AND METHODS

L A P purification. All reagents were of the highest purity commercially available. Leucine aminopeptidase was isolated from ox lens tissue by the procedure of Hanson et al. (1965). After two recrystallizations from ammonium sulfate the enzyme had a specific activity of >650 tLmole/min/mg (Carpenter and Vahl, 1973) in the magnesium-zinc form and appeared homogeneous in gel electrophoresis (Melbye and Carpenter, 1971). Electron microscopy. Electron micrographs of single molecules and crystal sections of leucine aminopeptidase were obtained on either Philips 300 or Siemens 1A electron microscopes operated at 80 kV and

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equipped with anticontamination devices. Calibration was achieved using a replica grating with 2160 lines/ mm. All photographs were taken on glass plates. Carbon stabilized Parlodion films on 400-mesh grids were used for most single-molecule work. An occasional grid of ultrathin carbon on holey parlodion-carbon was also employed in an attempt to enhance contrast of these specimens. Larger mesh grids were used to mount thin sections of the crystals. Three techniques of specimen preparation were used for visualization of single molecules. These involved sequential application of drops of stain and sample, sequential spraying of enzyme and stain, or simultaneous spraying of stain and enzyme. In some instances the LAP was crosslinked with dimethyl suberimidate in order to stabilize the specimen using the conditions for maximum crosslinking described by Carpenter and Harrington (1972). This preparation was used in a fashion similar to that used with the uncrosslinked enzyme. Micrographs which displayed the greatest detail of single molecules were obtained when the sample was stained with Millipore-ffltered 1% sodium phosphotungstate at pH 7. In our hands uranyl acetate and uranyl oxylate showed lower resolution. Crystal growth. Hexagonal needle crystals large enough for X-ray diffraction and for thin sectioning were grown at room temperature by vapor-phase equilibration, using 6% ammonium sulfate or 35-45% 2-methyl-2,4-pentanediol in 0.05 M Tris, pH 8.0, or 65% 2-methyl-2,4-pentanediol in 0.2 M triethanolamine, pH 8.0 (Jurnak et al., 1977). The organic precipitant gave larger crystals, with crystals with increased width to length ratio, than those obtained from ammonium sulfate. Spherulitic crystals similar to those mentioned by GlOsser et al. (1970) were obtained in early trials when precipitation was too rapid. Embedding and sectioning. In preparation for visualization in the electron microscope the crystals were rinsed twice in 0.2 M triethanolamine, pH 8.0, 25°C, crosslinked overnight with 0.1% glutaraldehyde, fixed with uranyl acetate, and embedded in Maraglas (Langer et al., 1975) using the technique of Erlandson {1964) as modified by Bensch (personal communication). The uranyl acetate treatment was omitted on some crystals. After trimming the plastic blocks, thin sections were cut on a Sorvall MT-2 Ultramicrotome equipped with a diamond knife. In order to check for compression more sections of the same plane were cut after rotating the crystal by 90 ° . The sections were lifted from the boat of the microtome, expanded by xylene or chlorofirm vapors, and mounted on grids. After sectioning some sections were re-treated with uranyl acetate and lead citrate (Reynolds, 1963) in order to enhance contrast. A procedure which facilitated the alignment of the crystals for thin sectioning involved embedding the crystal in a drop of Maraglas on a siliconized slide. Following polymerization each droplet became a plastic lens in which the edges of the crystal were clearly visible. These lenses were lifted

94

TAYLOR, CARPENTER, AND WLODAWER

from the slide and remounted on plastic posts in preparation for sectioning. Photographic prints. Prints of all micrographs were made on high-contrast paper. All photographic averaging was done using a Bessler CB7 enlarger with the emulsion side of the glass negative facing the film. Thin-section micrographs were averaged with a moving stage (Markham et al., 1964)while singlemolecules were superimposed by moving the negative. Rotary averaging (Markham et al., 1963)was done by rotating the stage. RESULTS Several preparations of LAP, each with specific activity of >700 /~mole/min/mg, were used for obtaining images of the single molecules in electron micrographs. All three m e t h o d s of sample application mentioned under Materials and Methods gave identical images. Some of the preparations were crosslinked with dimethyl suberimidate (Carpenter and Harrington, 1972) in an a t t e m p t to preserve structure during specimen spreading and electron imaging. While shapes of the crosslinked species were easier to categorize, they did not display b e t t e r retention of any specific form t h a n the noncrosslinked molecules. One thousand six h u n d r e d thirty-one images were categorized. A typical field is shown in Fig. la. Clearly definable equilateral triangles with sides ranging from 110 to 145 A with an average of 130 A made up 35% of the images. Of these 25% had another denser triangle, composed of three spheres, inset into the large triangle and rotated 60 ° relative to it (Fig. lb). T h e length of a side of this inner triangle ranged from 72 to 100 A and averaged 84 A; the diameter of each sphere was 40 A (range 28-51/~). In some of the individual micrographs as m u c h as 60% of the triangles showed this internal structure. Unclear triangles resembling the dense region alone

accounted for 5% of the images. Structures which appear tetrameric and which resemble views of glutamate decarboxylase (To, 1971) and glutamate dehydrogenase (Josephs, 1971) comprised 8% of the images (Fig. la). T h e s e had a long axis of approximately 135 A (range 123-145 A) and a short axis of 93 A (range 74-110 A), and the a p p a r e n t diameter of the m o n o m e r measured approximately 43/~. Two parallel arrays (Fig. lg) similar to images seen in Richards and Williams' (1972) type II preparations of aspartate carbamoyltransferase (ATC-ase) appeared 4% of the time. This percentage did not increase in more dense stain. T h e boundaries of such images were unclear. T h e y were approximately 95-130 /~ in length, 30-45 A in width with an interparticle space of 7-14 A. Smaller aggregates accounted for 11% of the images. Of the remaining images 16% were unclear views of the above structures and 21% could not be identified at all. In an a t t e m p t to increase the signal-tonoise ratio two types of photographic averaging were employed. A montage of five images showing the triangular outline was made (Fig. ld). This should average out noise, t h e r e b y emphasizing structure. T h e s e photographs imply t h a t the central triangle is composed of identical spherical substructures with diameter 41-46 A. T h e side of this triangle is 83 A and the side of the outer triangle is 124 A; thus it appears t h a t while the dense triangle is smaller t h a n the outer triangle, it is slightly larger t h a n what can be completely inscribed by the outer triangle. M a n y LAP images appear to have threefold s y m m e t r y but we chose not to eliminate a priori the possibility of pseudo sixfold symmetry. Molecules were rotated

FIG. 1. Fields ofLAP embedded in sodium phosphotungstate. (a) Field showing a predominance of triangular shapes (solid arrow) and some tetrameric shapes (dashed arrow). (b) Field showing predominance of a triangular structure which circumscribes a closed trimeric substructure. (c) Unprocessed image of one molecule selected from (b). (d) Photographic montage of the images of five particles selected from the micrograph shown in (b). (e) Image created by threefold rotary averaging through 120°. (f) Image created by rotary averaging six times through 60°. (g) An example of a parallal array. (a)-(g), × 370 000.

ELECTRON MICROSCOPY OF LEUCINE AMINOPEPTIDASE

95

i~, i,,~ ~~ii~ i~,~ij~%i

96

TAYLOR, CARPENTER, AND WLODAWER

FIG. 2. Electron micrograph of section cut perpendicular to the c axis of the crystal (0001) plane. Protein appears dark and solvent channels light. (a) Unprocessed image. (b) Image obtained after photographically averaging six times. × 600 000. both three and six times and by either 120 ° or 60 ° . Rotating three times by 120 ° produced the picture shown in Fig. l e and this is identical to the montage described above. T h r e e and sixfold rotations through 60 ° show images which are b o t h different from each other and different from anything else observed. T h e sixfold rotation (Fig. lf) shows the following images most clearly: an inner dense area with sixfold s y m m e t r y and 67-85/~ in diameter appears but it is circumscribed by an outer less dense region 110-130/~ in diameter with 60 ° offset. Several L A P crystals were used to obtain electron micrographs of thin sections cut parallel to (0001), (10i0), and (1120) planes. Greatest detail was visible in (0001) sections, probably due to the presence of protein-free solvent channels and dense protein zones (Fig. 2). T h e length of the a axis was calculated by measuring the distances between rows in this projection, directly on the micrograph plate. T h e distance calculated varied between 110 and 130 A and never exceeded 132/~, the value measured by X-ray diffraction (Jurnak et al., 1977). This result is not unexpected, since some

degree of shrinking is usually present in the d e h y d r a t i o n steps (Longley, 1967; McPherson and Rich, 1973). Photographic averaging of the (0001) projection enhanced the image (Fig. 2b) and clearly showed the presence of one large solvent channel per unit cell, as well as six denser spots in the corners of the cells, probably due to local superposition of particularly protein-rich regions. T h e s e images showed gross similarities but are different in detail from those observed by Kiselev et al. (1977) using thin crystals (rather t h a n sections of thick crystals). Electron micrographs of sections cut parallel to (1120) consisted of lines of alternating high and low density (Fig. 3). In these micrographs few details were observed in the direction of the (0001) axis. Contrast was low since there are no protein-free channels in this perspective. As expected, no contrast was present in the (10i0) micrograph. M e a s u r e m e n t of the unit cell parameters from the micrographs revealed values of a = 120 A (±10 A) of c = 110 A ( ± 1 5 / ~ ) , which correlate with the X-ray data. T h e unit cell p a r a m e t e r s and the ap-

ELECTRON MICROSCOPY OF LEUCINE AMINOPEPTIDASE

97

FIG. 3. Electron micrograph of section cut parallel to c axis of crystal (1130) plane. (a) Unprocessedimage. (b) Image obtained after photographicallyaveraging 10 times. × 600 000. pearance of the electron micrographs were similar for the crystals grown both from ammonium sulfate solutions and from 2methyl-2,4-pentanediol. While the unit cell parameters in stained and unstained specimen negatives were similar, the contrast apparent in the photographs was much greater for the stained preparations. Compression of up to 10% was observed during comparison of diffraction patterns of sections of the same plane cut perpendicular to each other. DISCUSSION The unit cell parameters for LAP crystals, as calculated from the X-ray diffraction data, are a = b = 132 A, c = 122/~, space group P6322 (Jurnak et al., 1977). These data indicate that there are two protein molecules per unit cell, located so as to fulfill the requirements of 32 symmetry (International Tables for X-ray Crystallography, p. 289). These X-ray diffraction data, along with chemical and physical data on t h e size of the subunits, set boundary conditions for interpreting the electron micrographs. Several configurations of the hexameric

enzyme, each of which fulfills the symmetry requirements and which employs spherical subunits, were proposed by Carpenter and Harrington (1972). One of these, the planar hexagon with heterologous interactions, was eliminated by crosslinking data. Another arrangement of spherical subunits, the octahedral array, was proposed by Damaschun et al. (1973). However, micrographs obtained by Wangermann et al. (1976) indicated that the two trimers were offset by less than 60 °. Based on a molecular weight of 54 000 and a partial specific volume of 0.74 cm3/g, each subunit would be about 50 /~ in diameter. While some micrographs (Figs. la,b,g) contain images similar in contour to those previously associated with hexameric enzymes with spherical subunits in octahedral array (To, 1971; Josephs, 1971), the dimensions and contrasts of the images and the results of photographic processing (Fig. lf), which are similar to that used previously (To, 1971) eliminate this possible arrangement. The majority of the clearest images (Figs. lb-e) cannot be reconciled with any of the models which are based on spherical protomers.

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TAYLOR, CARPENTER, AND WLODAWER

a

b

FIG. 4. (a-c) Photographsof proposed model. (a, c) Viewsalongtwofoldaxes. (b) Viewalong threefoldaxis. A configuration of the LAP molecule which is based on the images of single molecules (Figs. lb,c) but which assumes bilobal rather than spherical subunits is shown in Fig. 4. This model bears strong resemblance to the structure proposed for aspartate carbamoyltransferase (Richards and Williams, 1972; Wiley et al., 1971; Evans et al., 1973) and involves a bilobal shape for the subunit. The sides of the internal and external triangles of LAP are approximately 85 and 130/~ long, respectively. In this model the inner triangle is formed by the principal lobes, each with a diameter of 40-45 /~. The remaining protein radiates out at an angle to give the outer lobes which form the less dense outer triangle. Two trimers made of such nonspherical subunits are stacked on top of one another so that the dense inner parts are eclipsed when viewed down the threefold rotational axis. The outer triangle observed in the singlemolecule micrographs is formed by the outer lobes of each monomer. When viewed along the twofold molecular axis, some molecules could appear as parallel arrays. The molecule would be very unstable if placed on a grid in such manner that this view could be obtained; thus our inability to obtain micrographs showing a majority of the molecules viewed down the twofold axis is not unexpected. Images of tetrameric aggregates with a major axis of 123-145 ,~ and a minor axis of 74-110/~ can be reconciled with this view if only the subunits closest to the grid were embedded in stain. These views of the molecule would occur infre-

quently because the molecule would have to set down on its side in order to yield such projected images. The tetrameric appearing aggregates are most probably unequally embedded hexamers. Electron micrographs of thin sections of the crystalline enzyme are, in part, a result of the configuration of the enzyme. Sections cut perpendicular to ~he c axis of the crystal (Fig. 2) show light channels (approximately 60/~ diameter) in a dark honeycomb network. The distance between the channels, as measured directly on electron micrographs, varied between 110 and 130/~. This compares well with the unit cell parameters determined by X-ray crystallography (Jurnak et al., 1977) and it can be concluded that each unit cell has one solvent hole extending throughout the crystal lattice. A similar pattern was observed by Labaw and Olson (1970) for bacteriochlorophyll protein. These images were produced by the stacking of trimers located on special positions of the space group P63 (Fenna et al., 1974). The similarity of images of LAP, particularly after photographic averaging (Fig. 2b), to those of bacteriochlorophyll protein suggests that the packing of the two proteins might be similar. Further insight on the plausibility of various configurations of subunits in the polymer is obtained by considering the packing of molecules in the unit cell. The centers of both LAP molecules within a unit cell must occupy one of four special positions, either on the 63 (settings a or b) or on the threefold axes (settings c or d) (International Tables

ELECTRON MICROSCOPY OF LEUCINE AMINOPEPTIDASE Q /x / \

L r

A / \

"-¢.( 1;

i I

FIG. 5. Crystal packing diagram for models of LAP which assume bitobal subunits; with molecules placed on threefold axes. (a) The (0001) projection. Triangles inscribing circles represent outline of outer triangle mentioned in text. Circles represent inner more dense part of the images of single-molecule micrographs. Upper and lower molecules are indicated by solid and dashed circles, respectively. Four unit cells are outlined in boldface type. (b) The (1120) projection. Front and rear subunits are indicated by large and small circles and appendages, respectively. Two appendages in each molecule have been left out to increase clarity of unit cell cross section indicated in this projection. The unit cell is indicated by the straight lines in boldface type.

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graphs of the thin sections. This packing is shown in Fig. 5a. Only minimal collisions between molecules result if the central lobe is a 40-/~-diameter sphere, and the appendage is approximately 45 /~ long, has a tapering width of 20-35/~, and is 20/~ high along the c axis (Figs. 4a-c, 5b). All of the protein volume would be accounted for and a central hole 60/~ in diameter would result. T h e darkest spots in the micrograph (Fig. 2b) would result from those points where the appendages overlap the central spheres at a neighboring molecule. Molecules of this type can easily be packed into the unit cell in the less restrictive space group P321 proposed for microcrystals b y Kiselev e t al. (1977). While an unequivocal model for L A P and its crystal packing is not defined by the above data, A T C - a s e - t y p e organization of the molecule is indicated by the single-molecule images and results in a model which can be packed into the crystal so as to account for the appearance of the thin-section images. The authors gratefully acknowledgethe help of Dr. R. C. Williams, Alice Taylor, Irma Daehne, K. C. McFarland, and Dr. K. Bensch in obtaining electron micrographs. This work was supported by National Institutes of Health Grant EY00813. A.T. and A.W. were recipients of National Institutes of Health Research Service Awards 5-F02-54141-02 and 1-F32N505191, respectively. A preliminary report of this work appeared in (1977) Fed. Proc. 36, 809. REFERENCES

for X - r a y Crystallography, p. 289). Settings a and b differ only in the choice of origin along the c direction and are indistinguishable by electron microscopy. B o t h of these can be eliminated since t h e y would not yield images similar to those which we have observed. For the purpose of this analysis only setting c, where the centers of molecules are at (1/3, 2/3, 1/4) and (2/3, 1/3, 3/4), need be considered. Packing of the bilobal model of L A P into the crystal m a y be accomplished in several ways, b u t only one can be easily reconciled with the appearance of the electron micro-

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