J. Mol. Biol. (1976) 101, 185-196

The Structure of Actinidin E. N.

at 5-5 A Resolution

BAKER

Department of Chemistry, Biochemistry Massey University New Zealand

and Biophysics

(Received 25 July 1975) The structure of actinidin, a sulphydryl protease obtained from the fruit of Actinidia chkensis, has been determined from X-ray crystallographic data to a resolution of 5.5 A. Three isomorphous heavy atom derivatives, prepared with uranyl acetate, dichloroethylenediamine platinum(I1) and potassium iodomercurate(II), were used in the phase calculation, giving a mean figure of merit of 0%3. The molecule can be described as an oblate ellipsoid with approximate dimensions 50 d x 40 A x 36 A, and consists of two globular units separated by a shallow cleft. Binding studies with mercuric chloride reveal two sites of attachment, both within this cleft, and although both sites are of low occupancy it is probable that one or other marks the position of the active sulphydryl group. Although the folding of the polypeptide chain of actinidin cannot be followed with certainty, it appears to be closely similar to that of papain, suggesting that these are members of a family of homologous proteins.

1. Introduction Actinidin, a proteolytic enzyme obtained from the fruit of the Chinese gooseberry, Actinidia chinensis, has been shown (Arcus, 1959; McDowall, 1970) to contain a sulphydryl group essential for activity. It has therefore been grouped with a number of other sulphydryl proteases which have been obtained from a variety of plant sources, and which have been reviewed by Glazer & Smith (1971). These proteins show substantial similarity in their chemical and physical properties. Similar amino acid sequences about the active cysteine and histidine residues have been demonstrated for papain, kin and stem bromelain. Kinetic studies indicate a similar mode of action, and it has been suggested that plant sulphydryl proteases form a family of homologous proteins. Nevertheless, considerable variations are seen in their amino acid compositions, molecular weights, isoelectric points and in carbohydrate content. For example, actinidin has a molecular weight of approximately 26,000 (Boland & Hardman, 1972), compared with 23,400, 25,500 and 33,500 for papain, ficin and stem bromelain, respectively. The isoelectric point of actinidin is 3.1 (McDowall, 1970), very different from the values for papain (8*75), ficin ( >9) and stem bromelain (9.55). The amino acid composition of actinidin (A. Carne and C. H. Moore, personal communication) indicates that it contains only five Cys residues, compared with seven in papain, eight in kin and ten in stem bromelain. 185

186

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N.

BAKER

The present structure analysis was undertaken in order to compare the threedimensional structure of actinidin with that of papain, the most extensively characterized of the sulphydryl proteases, and the only one whose three-dimensional structure has so far been determined (Drenth et al.: 1971). This will establish Tvhether or not the two proteins are closely sin&r in structure as well as function, and \vill enable a detailed analysis of the differences between them to be made.

2. Materials (a)

and Methods and

Crystallization

crystal

properties

The purification and crystallization of actinidin have been reported previously (Baker, with cell dimensions a=78.2A, b=81.gLk, c:= 1973). The crystals are orthorhombic, 33-O A, space group P212,2,, with one molecule of actinidin per asymmetric unit). Most preparations yielded either small single crystals or aggregates of larger crystals, (xx-en though many variations on the crystallization conditions were tried. The best results were obtained by dialysing a 10 mg per ml protein solution against a 20 to 25% saturated ammonium sulphate solution, maintained at pH 6.0 with 0.1 M-sodium phosphate. Large plate-like crystals could often be dislodged from the aggregates obtained. The crystals used were obtained initially from solutions of the tetrathionate-inactivated enzyme. Inactivation with tetrathionate is reversible, and in an attempt to facilitate t,he binding of heavy atom compounds at the active site, the crystals were reactivated b> soaking in standard mother liquor (25% saturated ammonium sulphate solution, pH 6.0) containing 0.05 M-dithioerythritol. Several changes of this solution were made over a period of two days, before the crystals were re-equilibrated with standard mother liquor. This procedure appeared to unblock the active site, in that it allowed some binding b> mercury compounds. The native crystals were also treated in this way before data collection. (b)

Heavy

atom

derivatives

All derivatives were prepared by soaking the crystals at 4°C in solutions of the st,andard mother liquor to which the heavy atom compounds had been added. The two most promising derivatives were many1 acetato and Pten(!l,t. \Vit,h umnyl acetate substantial intensity changes in the diffraction pattern were seen, wit’h cell dimension changes of less than 0.3%. Intensity changes appeared to be the same for uranyl acetate concentrations of 5 mM or 10 rnM and soaking times ranging between two days and four weeks. For PtenCl, a saturated solut,ion was used, and again the crystals showed significant intensity differences, with good isomorphism. Soaking times of 1 t,o 3 weeks produced apparently identical changes. Diffraction changes were also obtained after soaking crystals in 5 mx solutions of potassium iodomercurate(II), K,HgI,, and potassium t’etrachloroplatinit,e, K2PtCl,, although these were more sensitive to concentration and t,he soaking time. A 2.5 IIIM solution of sodium chloroaurate(III), NaAuCl,, caused actinidin crystals to turn light orange in colour and produced changes in their diffraction pattern. This derivative was not investigated further, however, because after mounting in a glass capillary tube the crystals continued to change colour, to a deep red, and the diffraction pattern changed with time. Attempts were also made to prepare derivatives in which a mercury atom was attached to the active sulphydryl group. When tetrathionate-inactivated crystals were soaked in solutions of mercury compounds no diffraction changes were observed. Crystals which had been previously treated with dithioerythritol (as described above) did show intensity changes when soaked in 5 mM-mercuric chloride and in saturated solutions of ethylmercury chloride and methylmercury iodide. The changes were slight, however, suggesting either low reactivity of the sulphydryl group in the crystals, or limited access to it. Attempts t Abbreviation

used:

PtenCl,,

dichloroethylenediamine

plat,inum(II).

STRUCTURE

OF

ACTINIDIN

187

to crystallize actinidin after previously reacting it with far been successful. Derivatives for which data were collected were : (1)

5 mM-many1 acetate, pH

(2) (3)

Saturated weeks. 5 mM-K,HgI,,

(4)

5

(5)

5 mlsl-K,PtCl,,

mM-InerCLlriC

acetate, in 25% 5.5, soaking time PtenCl,,

in

saturated 1 week.

standard

in standard chloride,

mother

in standard

(NH,),SO,

mother

mother

liquor

liquor,

in standard

liquor,

compounds

solution (phosphate

soaking

mother

(c) X-ray

mercury

time

liquor, soaking

buffered buffer),

with soaking

not

so

sodium time

3

1 week.

soaking time

have

time

5 days.

1 week.

data collection

Intensity data were collected at room temperature, with nickel-filtered Cu-Ka radiation ou a Hilger and Watts automatic 4-circle diffractometer. Integrated intensities were measured by an w-scan through the peak position, snd corrected for background by stationary measurements of the scattering on each side of the peak. For each crystal --reflections hkl and hkl were measured. In order to minimize absorption effects, each crystal was cut with a scalpel blade to a more cylindrical shape (typical dimensions 1.0 mm x 0.4 mm x 0.2 mm) snd mounted with its longest dimension along the axis of a Lindemann glass capillary tube. Absorption corrections were made using the semiempirical procedure of North et al. (1968). Peak to trough ratios for the absorption curves ranged from 1.5 to 2.5. The crystals showed considerable resistance to radiation damage. An entire native -_2.8 A data set (11,600 reflections, including hkl and hkl) was collected on a single crystal. Over the period of 2 weeks required for the data collection, 3 standard reflections (measured every 100 reflections) showed a drop in intensity of only 6%. The merge index R, for rodundent measurements of the intensities of symmetry-related reflections in the 2.8 A n d&a set was 0.638 (where R,= 2 ( 2 IZi-ll)/C nf). The stability of the heavy atomhkl i-1 hkl subst,ituted crystals is not known, since low resolution data only has been collected for them. However, no significant decrease in intensity was noted for any of them during the 2 to 3 days required to collect’ low resolution data. 4.5 A data were collected for the uranyl acetate and PtcnCl, derivatives, and 5.5 A data for the K,HgI,, K,PtCl, and HgCl,-soaked cryst,als.

3. Results and Discussion (a) Location

and refinement

of heavy atom sites

Initial heavy atom sites for each derivative were found by using the isomorphous differences in the calculation of three-dimensional (dF)2 difference Patterson syntheses (Blow, 1958). The three Harker sections (space group P2,2,2,) for each of the derivatives used in calculating the 5.5 d electron density map, vii. uranyl acetate, PtenCl, and K,HgI,, are shown in Figures 1 to 3. Each was initially interpretable in terms of two major heavy atom sites, although interpretation of the K,HgI, difference Patterson map was rendered more difficult by the presence of cross-vectors on the sections UV$ and ivw. Each derivative was refined separately, by means of a full matrix least-squares program based on ORFLS (program developed by Busing, Martin and Levy, of Oak Ridge National Laboratory, Oak Ridge, Tennessee). The quantity minimized where Id F, 1 is the observed isomorphous difference (/BP, 1 = was CC PO1 -M2, lI~PHI-I~pII) and f~ is the calculated heavy atom structure amplitude. Centric data only was used, the data from the three centric projections being combined to give a pseudo-three-dimensional set of centrosymmetric data. Occupancies and

188

E.

N.

BAKER 0.5

0

Y

,

0.5

b03

u

Q

0

o0.5

k

u=@5

FIG. 1. The 3 Harker sections of the (dF)2 Patterson map for the uranyl acetate derivative of actinidin. Contour levels are 3%, 5%, go/o, 12% etc. of the origin peak, with negative contours omitted. Self-vectors (i.e. vectors between atoms related by crystallographic symmetry elements) for the 2 major sites are indicated with ( n ). Cross-vectors which accidentally appear on, or close to, a Harker section are marked with a cross.

x, y and z atomic co-ordinates were refined, but because the data were of low resolution, temperature factors were fixed at 20 A2 for the many1 acetate and PtenCl, derivatives, and 70 Aa for the K,HgI, derivative. A trial calculation showed that there was little tendency for the temperature factors to refine away from these values. Initial refinement of the two-site models for each of the derivatives gave centric R values (see Table 1 for definition) of R, = 0.47, 0.47 and 0.51 for the uranyl acetate,

STRUCTURE

OF

ACTINIDIN

189

W

(

,5

C

3.5

0h

09

v u

V

0

0 X

0,

v=o.5

O.!

uao.5

FIG. 2. The 3 Harker sections of the (dP)2 Patterson map for the PtenCl, derivative of actinidin. Contour levels and the designation of peaks are as in Fig. 1. The elongation of the peak enclosing the 2 major self-vectors in section (WV&) can be explained by the presence of a, third (minor) site of substitution (see t!ext).

PtenCl, and K2HgI, derivatives, respectively. Three-dimensional difference Fourier syntheses were then examined in order to identify possible minor sites of heavy atom substitution. The uranyl acetate derivative (for which anomalous scattering was the most pronounced) was used to calculate preliminary protein phases with both isomorphous and anomalous differences being used in the phase calculation (Matthews, 1966). Two sets of phases were calculated, corresponding to the two possible hands for the uranyl heavy atom configuration; one gave much cleaner difference Fourier

190

E.

N.

BAKER

0

v=o5

03

ll=O.5

FIG. 3. The 3 Harker sections of the (dP)2 Patterson map for the HgI:Contour levels and the identification of peaks are as in Fig. 1.

derivative

of actinidin.

maps for both PtenCl, and K,HgI, derivatives, and peaks which corresponded very well with those expected from the original difference Patterson maps. This choice of hand was therefore taken as the correct one. The difference Fourier synthesis for the KsHgI, derivative revealed one minor site. That for the platinum derivative contained no clearly-defined minor sites, but one of the major peaks (site A) was noticeably elongated. The introduction of a third site (of about one-third the occupancy of the main site, A) to account for this then produced a significant decrease in the residual R,. A difference Fourier for the uranyl acetate derivative

STRUCTURE

OF

ACTINIDIN

TABLE

191

1

Heavy-atom FrameteW and phasing statisticsb Fractional Derivative

uog+ PtenCl,

HgI&-

HgCl,

Site A B c A B c: A B C A B

Occupancy (electrons) 41 36 14 33 28 11 46 46 14 19 19

co-ordinates

32 0.220 0.257 0.077 0.187 0.286 0.161 0.238 0.222 0.517 0.287 0.258

a Temperature parameters, B, were 20 A2 for and 70 As for HgI2,sites. b Mean figures of merit for centric, non-centric The HgCls derivative was not included in the “Rc = ;llh, - PPI -fell ~1% - PPI

Y 0.164 0.199 0.116 0.299 0.225 0.335 0.288 0.490 0.299 0.236 0.292

z 0.868 0.823 0.026 0.689 0.539 0.650 0.646 0.917 0.094 0.576 0.626

all sites in UO:

RCC

r.m.s.

fHa

r.m.s. E"

0.42

95

42

0.42

78

26

0.47

94

49

0.50

+, PtenCl,

and HgCl,

derivatives

and total data are 0.94,0.86 and 0.88, respectively. phase calculation. calculated for centric reflections only, where

P,,

is the structure factor of the derivative, F, that for the protein, andfa for the heavy atoms. d r.m.s. fa = [zfi/N] t where fh is the heavy atom scattering amplitude for reflection h and N is the number of reflections. e r.m.s. E = [z&/N]* where < is the lack of closure at the most probable phase for reflection h of derivative

jfand

N is the number

of reflections.

(phased by PtenCl,) also revealed a third site of substitution. Confirmation of the minor sites in all three derivatives was given by the identification of the expected cross-vectors (with the major sites) in the original difference Patterson maps. Refinement of these three-site models for the uranyl acetate, PtenCl, and K,HgI, heavy atom derivatives finally gave R values, for the centric data, of O-42, 0.42 and 0.47, respectively. Of the other heavy atom derivatives, the difference Patterson map for PtClishowed peaks coincident with those of the two major sites of PtenCl,, but also contained a number of other peaks which could not be accounted for. The latter may have arisen from a lack of isomorphism. The difference Patterson map for HgCI, indicated two sites, which were subsequently confirmed by a difference Fourier synthesis phased by the uranyl and PtenCl, derivatives. Both sites were of low occupancy, however, and least-squaresrefinement returned a reliability factor, R,, of 0.50. The uranyl and PtenCl, derivatives appear to be very suitable for a high resolution structure analysis, and 2.8 ,& data are currently being collected. K,HgI, was employed as a third derivative in the calculation of a low resolution (5.5 8) protein Fourier synthesis, but its greater sensitivity to concentration and soaking time, and the probable greater complexity of the heavy atom configuration, may make it lesssuitable for the high resolution analysis. Heavy atom and phasing parameters are given in Table 1.

192

E.

N.

BAKER

(b) Electron density map and model Structure factors were placed on an approximately absolute scale by means of a Wilson plot (Wilson, 1949) using the native 2.8 A data set. The three best heavy atom derivatives, viz. uranyl acetate, PtenCl, and HgIi-, were used to calculate phases for the native protein data, by the method of Blow & Crick (1959). Anomalous scattering measurements for each derivative were also taken into account (Matthews. 1966). Values of E, (the root-mean-square estimate of the error in isomorphous differences for the jth derivative) were taken as the root-mean-square lack of closure of the isomorphous replacement triangle for that derivative (see Table 1). Values of E; (the root-mean-square error in the anomalous differences for the jth derivative) were taken as the root-mean-square difference between the observed and calculated anomalous differences. The ratios E/r.m.s. fH were 0.44, 0.33 and 0.52 for the uranyl, PtenCl, and HgIzderivatives, respectively, and were essentially constant over all sine/h values within the 55 A sphere. The mean figure of merit for the 780 reflections with spacings greater than 5.5 A was 0.88. The protein electron density map was calculated from structure factors weighted by the figure of merit, WL The maximum electron density was found to be 0.35 electrons per A3 (no F,,, term was included) and was contoured at equal intervals of about 0.05 electrons/A3, starting from the lowest contour of 0.09 electrons/A3. The molecular boundary was extremely well defined, there being only two points at which electron density from one molecule coalesced with density from a neighbouring molecule (see Fig. 4). A balsa-wood model, representing a single molecule, was therefore constructed, to include all density greater than 0.07 electrons/A3. Two views of the model are shown in Figure 5(a) and (b). The molecule is an oblate ellipsoid of approximate dimensions 50 A x 40 A x 36 A, and has a double domain appearance with the two globular units being separated by a shallow cleft. In these respects actinidin clearly resembles papain (Drenth et al., 1967). The cleft contains both binding sites for the enzyme inhibitor, HgCl, which binds at two positions approximately 6 A apart. Although both are of low occupancy (about 25%)> it seems probable that this cleft, as in papain, contains the active site of the enzyme, and that one or other of the HgCl, binding sites marks the position of the active sulphydryl group. The observation that the overall size and shape of the actinidin molecule are very similar to that of papain encouraged a closer examination of the electron densit’y map. The most striking feature is a dense rod of electron density, about 24 A in length, running through the centre of the molecule. It begins directly beneath the “active site” cleft, within about 5 A of the two mercury-binding sites, and forms part of bhe interface between the two halves of the molecule. Both for its size and locat’ion? this feature was immediately suggestive of the central five-turn a-helix in papain (residues 24 to 41). In papain the active sulphydryl group is at residue 25. and the helix (24 to 41) forms part of the wall of one of the two domains of the papain molecule, at the interface between the two (Drenth et al., 1971). An attempt was then made to follow the course of the polypeptide chain through the electron density map. As expected, this could not be done unambiguously. because of the numerous intersections between parts of the chain. When it was assumed, however, that the central rod of density (mentioned above) could be identified with the a-helix 24 to 41 in papain, and that the overall pattern of folding was likely to be similar, a satisfactory fit to the observed electron density could be achieved. In many parts, the chain could be followed unambiguously without

STHUUTURE

OF

ANTlNIUIN

193

Pm. 4. Photograph of a portion of the 6.5 A resolution electron density m8p of actinidin. The positive z direction runs from left to right. 10 sections are superimposed, from z = 0.53 to z = l-06, The probable molecular boundary is shown with a dotted line. The solid arrow indicates a dense rod of electron density slanting downwards towards the “bottom” of the molecule, and corresponding inlength and position to the central a-helix (26 to 41) in papain. The 2 HgCl,-binding sites are close to the arrow head. Two other possible a-helices are marked with hollow arrows. The one on the left of the molecule runs vertically (parallel to z axis) and probably corresponds to the helix (116 to 126) in papain. The one at the top of the photogmph may correspond to 69 to 78 in papain. Other features which appear to correspond well with the structure of papain are marked by N (probable N-terminal end of the polypeptide chain); C (probable C-terminal end); and B (a protruding piece of density analogous to the hairpin p-loop in papain 164 to 172).

reference to the papain structure. This was especially true in domain 1 of the molecule (see Fig. 5), which corresponds to that part of the structure designated lobe L in papain, and which, in the latter, contains residues 11 to 111 and 209 to 212. The three sections of chain which cross from domain 1 to domain 2 are also clearly defined, but the folding in domain 2 was much more difficult to follow. In papain, this part of the molecule has a twisted @-sheet structure. Where density from several sections of chain coalesced, the decision on which direction to take was based on the papain

FIG. 5. Two views of a balsa-wood model of actinidin. (a) Viewed approximately --z direction. The probable active site cleft is at the top of the molecule, separating (on right) from domain 2 (on left.). (b) Viewed from on “top” (i.e. looking down +z Domain 1 is on right, domain 2 on left. The approximate course of the cleft between and 2 is marked with a broken line. Major heavy atom binding sites are marked with cross-hatched discs to indicate the 2 HgCl, binding sites. Numbers identify the sites 1, HgCl, site A and PtenCl, site B; 2, HgCl, site B and HgIYsite A; 3, Uranyl sites 4, PtenCl, sites A and C; B, HgI:site B.

along the domain 1 direction). domains 1 discs, with as follows: A and B;

STRUCTURE

OF

ACTINIDIN

195

structure and the necessity to include all the electron density in a continuous folding scheme. Only two places were encountered where density not present in the map was required, and in both cases the density, although below the minimum contour level, was still positive. Apart from a few small isolated features at the lowest contour level, and some lobes of density (perhaps side chains) projecting from the main chain, no electron density in the area of the molecule is left unexplained. As well as the central u-helix, described above, two other dense rods of electron density correlated well with a-helices in papain. One, which has its axis parallel to the crystal c axis, corresponds to the a-helix 116 to 126 in papain, and is clearly seen in Figure 4. The other, also indicated in Figure 4, corresponds to the a-helix 69 to 78 in papain. Another prominent feature is a piece of electron density projecting from domain 2 into the solution. It probably corresponds to the hairpin loop of p-structure, 164 to 170 in papain, and is adjacent to the NH,-terminal end of the protein. Although papain has three disulphide bridges and actinidin probably only two, it is not possible to tell, from the low resolution map, which of them is missing. In each of the expected positions the chains come close together and there is connecting density between them. It was felt that the number of residues along the polypeptide chain could not be accurately counted at this resolution. However, no major additions or deletions from the papain structure could be detected, apart from the possibility of a few additional amino acid residues at the COOH-terminus. The heavy atom binding sites are all seen to be on the surface of the molecule. The two major uranyl sites, which are about 5 A apart, lie on either side of a piece of density extending from the main chain at the top of the molecule. Site B for PtenCl, is coincident with one of the two HgCI, sites in the “active site” cleft while the remaining HgCl, site, sites A and C for PtenCl, and one HgI$- site are also in the same general area. These positions are indicated in Figure 5(b). The present interpretation of the low resolution electron density map for actinidin can only be regarded as tentative, and must await confirmation from an analysis at higher resolution. Nevertheless, it is clear that actinidin and papain have a number of structural features in common, and that the actinidin electron density map is at least consistent with similar folding of the polypeptide chains in the two proteins. This, therefore, supports the suggestion that the plant sulphydryl proteases make up a family of homologous proteins, similar in structure as well as function. The extension of the X-ray analysis of actinidin to a resolution of 2.8 J$ is well advanced, and will allow a detailed analysis of the similarities and differences between actinidin and papain, and their consequences for structure and function, to be made. I thank Dr P. P. Williams and Dr K. L. Brown of the Chemistry Division of the Department of Scientific and Industrial Research, Wellington, for their most generous assistance in making their 4-circle diffractometer available, and in helping with the data collection. I also thank Dr J. Drenth of the Department of Structural Chemistry, University of Groningen, The Netherlands, for communication of the atomic co-ordinates for papain, Drs S. V. Rumba11 and D. A. D. Parry for helpful discussions, Dr C. H. Moore and Mr A. Came for communication of their unpublished results, and other members of this department for their constant encouragement and interest in this work. REFERENCES Arcus, Baker, Blow, 14

A. C. (1959). E. N. (1973). D. M. (1958).

Biochim. Biophys. Actu, 33, J. Mol. Biol. 74, 411-412. Proc. Roy. Sot. 247, 302-336.

242-244.

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Blow, D. M. & Crick, F. H. C. (1959). Acta Cryetallogr. 12, 794-802. Boland, M. J. & Hardman, M. J. (1972). FEBS Letters, 27, 282-284. Drenth, J., Jansonius, J. N. & Wolthers, B. G. (1967). J. Mol. Biol. 24, 44%457. Drenth, J., Jansonius, J. N., Koekoek, R. & Wolthers, B. G. (19’71). Advan. Protein Chem. 25, 79-115. Glazer, A. N. & Smith, E. L. (1971). In The Enzymes (P. D. Bayer, cd.), vol. 3, pp. 501-546. Academic Press, New York. Matthews, B. W. (1966). Acta Crystallogr. 20, 82.-86. McDowall, M. A. (1970). Eur. J. Biochem. 14, 214-221. North, A. C. T., Phillips, D. C. & Mathews, F. 6. (1968). Acta Crystallogr. ser. A, 24, 351-359. Wilson, A. J. C. (1949). Acta Crystallogr. 2, 318-321.

The structure of actinidin at 5-5 A resolution.

J. Mol. Biol. (1976) 101, 185-196 The Structure of Actinidin E. N. at 5-5 A Resolution BAKER Department of Chemistry, Biochemistry Massey Universi...
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