Cocrystals of yeast cytochrome c peroxidase and horse heart ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 29. Issue of October 15, pp. 13881-13884,1987 Printed in U.S.A.

Communication Cocrystals of Yeast Cytochrome c Peroxidase and Horse Heart Cytochrome c* (Received for publication, April 27, 1987)

Thomas L. Poulos$g, Steven Sheriffll, and Andrew J. Howard$ From the $Protein Engineering Department, Genex Corporation, Gaithersburg, Maryland 20877 and the TNatwnul Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

inunderstandingprotein-proteininteractions and mechanisms of interprotein electron transfer reactions. We were very encouraged that thegoal of determining the x-ray structure of the complex might be realized once we succeeded in cocrystallizing the peroxidase and horse heart cytochrome c under conditionswhich should favor stabilization of the complex. However, as we describe in this report, the crystals consist of peroxidase dimers with orientationally disordered cytochrome c molecules trapped in thecrystalline lattice. MATERIALSANDMETHODS

Yeast cytochrome c peroxidase and horse heart cytochrome c have been cocrystallized in a form suitable €or x-ray diffTaction studies and the structure determined at 3.3 A. The asymmetric unit contains a dimer of the peroxidase which was oriented and positioned in the unit cellusing molecular replacement techniques. Similar attempts to locate the cytochrome c molecules were unsuccessful. The peroxidase dimer model was subjected to eight rounds of restrained parameters leastsquares refinement afterwhichthe crystallographic R factor was 0.27 at 3.3 A. Examination of a 2Fo-Fc electron density map showed large “empty” regions between peroxidase dimers with no indication of cytochrome c molecules. Electrophoretic analysis of the crystals demonstrated the presence of the peroxidase and cytochrome c in an approximate equal molar ratio. Therefore, while cytochrome c molecules arepresent in the unit cell they areorientationally disordered and occupy the space between peroxidase dimers.

Biological electron transfer reactions require the formation of intermolecular complexes between electron donor and acceptor protein molecules. Some of the more thoroughly studied electron transfer reactions are those involving cytochrome c (Salemme, 1977; Ferguson-Miller et al., 1979). Using known crystal structures, computer graphics modeling has been employed to develop hypothetical models for cytochrome c electron transfer complexes (Salemme, 1977; Simondsen et at., 1982; Poulos and Finzel, 1984). The cytochrome c peroxidasecytochrome c complex represents the only detailed model of a physiologically important complex involving cytochrome c (Poulos and Finzel, 1984). The peroxidase-cytochrome c complex has been investigated in several laboratories and currently is one of the most actively studied electron transfer systems (Bechtold and Bosshard, 1985; Bisson and Capaldi, 1981; Cheung et al., 1986; Liang et al., 1987; Waldemeyer et al., 1982; Waldemeyer and Bosshard, 1985). The crystal structure of the complex would provide important new information

* This work wassupported inpart by National Institutes of Health Grant GM 33688.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 Present address: Dept. of Chemistry/Biochemistry, University of Maryland and Center for Advanced Research in Biotechnology, 9600 Gudelsky Dr., Rockville, MD 20850.

Crystallization-Cytochrome c peroxidase was purified from commercial bakers’ yeast according to Nelson et al. (1977) and twice crystallized by dialysis against distilled water. Sigma type VI horse heart cytochrome c was dissolved in distilled water, oxidized with ferricyanide, and chromatographed over a Sephadex G-75 column equilibrated with distilled water. The middle two-thirds of the cytochrome c band were collected. 1:l molar ratios of the peroxidase and cytochrome c were combined to a concentration of 0.1 mM followed by exhaustive dialysis against distilled water. After dialysis, the complex was concentrated to about 0.4mM. Buffers were excluded from the crystallization medium to encourage electrostatic complex formation. Crystallization was achieved using the hanging drop vapor diffusion method. 5 microliters of complex were combined with 5 r l of 20% methylpentanediol and vapor diffused against 0.5-1.0mlof20% methylpentanediol a t room temperature. Crystals (Fig. 1) appeared within 24 h and grew up to 0.5 mm and occasionally up to 1 mm. We also have succeeded in cocrystallizing the peroxidase with yeast iso1-cytochrome c, but these crystals were too thin for crystallographic studies. Characterization of the Crystals-X-ray intensity data were collected with a Nicolet-Xentronics imaging proportional counter and a rotating anode x-ray source operating at 2.8 kilowatts. The unit cell dimensions and space group were determined directly from data obtained with the area detector as described by Gilliland et al. (1987). The autoindexing routine of the REFINE program in the XENGEN system’ was used to determine the unit cell parameters and were found to be a = b = 105.2 A and c = 186.6 A and a = B = y = 90”. These results indicated a tetragonal space group. Examination of individual frames of data with the aid of a TV monitor showed a 4fold screw axis perpendicular to a 2-fold screw axis demonstrating that thespace group must be P41212 or P43212.Assuming a molecular mass of 46,420 (34,030 for the peroxidase plus 12,354 for the cytochrome c) and two molecules of the complex/asymmetric unit, the Matthews coefficient, V , (Matthews, 1968), is 2.78 A3/dalton. The crystal used for obtaining the initial data set for space group determination was removed from the capillary, dissolved in 1%sodium dodecyl sulfate, and analyzed electrophoretically. As shown in Fig. 2, the crystal gave two bands corresponding to the peroxidase and cytochrome c. A sample of a 1:l mix of the two proteins was run on the same gel for comparison which indicates that thecrystal also contained a 1:l ratio of the two proteins. In subsequent experiments, gels of the crystals were scanned and gave a peroxidase:cytochrome c ratio of 1.24. Data Collection-A data set partially complete to 3.3 A was obtained from single crystal. A total of 84,187 observations of which 13,481 were unique scaled to give asnR,,, on intensities *= 0.098. The data set was 99% complete to 3.5 A and in the 3.5-3.3-A range, 35% complete. In the outermost shell of data between 3.5 and 3.3 A, the mean value of the ratio of the intensity to standard deviation was 2.05. Molecular Replacement and Refinement-Molecular replacement was carried out using the MERLOT package of programs (Fitzgerald, 1987). The refined peroxidase (Finzel et al., 1984) and tuna cytochrome c (Takano and Dickerson, 1984) models, both of which are available from the Protein Data Bank (Bernstein et al., 1977), were

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A. J. Howard, unpublished data.

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Crystals

Peroxidase-Cytochrome c package of programs (Hendrickson and Konnert, 1980; Hendrickson, 1985), and Table I lists the refinement statistics. The final R factor after eight cycles of refinement- with no manual interventions to adjust the model was 0.27 at 3.3 A. RESULTSANDDISCUSSION

Fig. 3 shows several sections of t h e 3.3-A 2Fo-Fc electron density map obtained after refinement of the peroxidase dimer. What is most striking about this map is the large "empty" of a regionsbetweenperoxidasemolecules.Somevestige cytochrome c molecule should have appeared in 2Fo-Fc, FoFc, or Bijvoet difference electron density mapsif one or more the cytochrome c moleculesoccupieddefinitepositionsin asymmetric unit. Bijvoet difference Fouriers should be particularly sensitive to locating iron atoms. As a control to test the quality of the iron anomalous signal, the peroxidase iron FIG. 1. Photograph of theperoxidase-horseheartcytoatoms were removed from the model and a set of Fc (no iron) chrome c cocrystals as they appear in the vapor diffusion well. phases computed. The resulting Bijvoet difference map The maximum dimension of a crystal shown is ahout 0.4 mm. showed the peroxidase iron atoms as strong peaks with the

TABLE I Summary of refinement least squares statistics rms deviation indicates the root mean square deviation of the final modelfrom ideal bond distances and angles expected from small molecule crystal structures. The target rms represents an adjustable target root mean square deviation used as a weighting parameter during the restrained least-squares refinement. 10-3.3 A Resolution range Reflections measured 13,481 Reflections used ( I > u n 8,999 R factor IFo-Fc I /CFo 0.27 Distances type

FIG. 2. Sodium dodecyl sulfate gel pattern of the cocrystal used for obtaining the initial x-ray intensity data set. Panels A and E are molecular weight standards. Panels B. C, and F are increasing amounts of a 1:l mix of the two proteins while panel D is the pattern of the single crystal. used as probes. We expected the asymmetric unit to contain two peroxidase molecules andthus, two peaks should appear in the rotation function using the peroxidase model as a probe. Indeed, two peaks at 7.2 and 5.2 u were observed using the Crowther rotation function (Crowther, 1972) with data between 10.0 and 4.0 A and a 24-A radius of integration. The solution was relatively insensitive to the radius of integration used in the bange 20-30 A but was sensitive to resolution. Peaks in the 10.0-6.0-A range were closer to 3-4 u and were considerably more difficult to distinguish from the noise level than the clearly defined peaks obtained using the higher resolution data. The Eulerian angles thus obtained were refined using the rotation function described by Lattman and Love (1972). In this case, the rotation search consisted of 9 steps in 2" increments around each of the three Eulerian angles obtained from the Crowther rotation function. Next, each molecule was positioned in the unit cell using the translation function (Crow$her and Blow, 1967). The search was made in increments of 1.05 A along the a and b axes and 0.93 A along c. For both peroxidase molecules in the dimer, only one peak between 6 and 7 u appeared on each of the three Harker sections giving a clean and unambiguous solution. A translation search between the two molecules in the asymmetric unit also gave a unique solution and allowed for the relative positioning of each of the molecules. Finally, the program RMINIM was used to refine the orientation and translation parameters and select the correct space group. The R value fell nearly 10 points to 0.45 when P43212was used as opposed to P4,2,2 so the correct space group was the former. All attempts to locate the cytochrome c molecules using similar procedures were unsuccessful. The oeroxidase dimer was refined usine the Hendrickson-Konnert

Through bonds Through bond angles Throughdihedral angles

rms deviation from ideality of final refined model

target rms

deviation

A

A

0.012 0.020

0.030 0.040 0.050

0.018

FIG. 3. Several sections of the final 3.3 A ZFo-Fc electron density map. The view is along the 4-fold ( 2 )axis with one monomer

Peroxidase-Cytochrome c Crystals

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FIG. 4. Stereoscopic diagram of the hypothetical peroxidase-cytochrome c complex according to Poulos and Finzel (1984).The peroxidase is on the bottom and cytochrome c on the top. Critical carboxylate residues inthe peroxidase andlysine residues in cytochrome c postulated to be involved in forming the electrostatic complex are labeled.

iron atom of molecule A of the peroxidase dimer appearing monomer, molecule B, because of the location of symmetry 5.8 times above background. Thus, the anomalous signalwas related peroxidase molecules. Unfortunately, these results do sufficiently strong to have enabledlocation of the cytochrome not allow us to draw any firmconclusions on whether or not c iron atoms. Since the electrophoretic analysis of the crystals the hypothetical model is a valid representation of the “true” demonstrated the presence of cytochrome c in an approximate electron transfercomplex. Finally, we consider two possible sources for the observed equal molar ratio to the peroxidase, we must conclude that disorder of cytochrome c: one static and one dynamic. The the cytochrome c molecules are present but orientationally disordered andoccupy the channelsbetween peroxidase mol- discussion that follows is very similar to thatof Maraquart et al. (1980) who found that the entire Fc region in crystals of ecules. Why did the peroxidase crystallize rather than a specific an intact antibodywas disordered. If there were two distinct orientations of the cytochrome c molecules, we should have and unique 1:l complex? At the very least, one might have expected thata stable 1:l complex should haveprevented the been able to observe some residual electron density for each peroxidase alone from crystallizing. By excluding buffers and of the orientations. It might also be possible to distinguish three orientations, but we doubt that more than three could using an organic solvent as a precipitating agent, the extremely low ionic strength should have promoted complex be distinguished with the available 3.3-A data. Therefore, if formation. The equilibrium association constant is strongly thereare four or more orientations of the cytochrome c dependent on ionic strength decreasing from 6 x lo6 M” at mvlecules relative to the peroxidase molecules that differ by 0.01 M salt to2.2 X lo3 M” a t 0.20 M salt (Erman andVitello, 2 A or more, we would expect the electron density correspond1980). Extrapolation to the ionic strength used for crystalli- ing to the location of the cytochrome c to appear as “flat” solvent as it does in our maps. The second possible source of 107-108 M” zationsuggests a n associationconstantnear be (Erman and Vitello,1980). Nevertheless,crystallization of disorderis dynamic.Dynamic disordercansometimes the peroxidase was favored(thermodynamically? kinetically?) semi-quantitated withcrystallographic B or temperature factors. A temperature factor .Of 80 A’ corresponds to a mean over complex formation. Next, we address the following question: is the surface of amplitude of vibJation of 1A and an87% decrease in scatterthe peroxidase that the hypotheticalmodel shows interacting ing power at 5 A resolution with essentially no contribution with cytochrome c available for interaction with orientation- to the diffraction pattern at 3A resolution. Temperature factors thislarge for protein and especially solvent atoms are ally disordered cytochrome c molecules in the crystal? The answer is yes. One molecule of the peroxidase dimer, desig- often very difficultto correctly model and usually are excluded nated molecule A, is oriented such that the surface we have from highresolution (1.5-2.0 A) refined structures. Theretore, postulated participatesin complexformation withcytochrome we would expect that a thermalvibration of 2 to 3 A is c (Fig. 4) and is available for interaction with the cytochrome. sufficient to prevent the cytochrome c molecules from conModeling of the hypothetical complexshows that a cyto- tributing significantly to the diffraction patterneven at very between low resolution. Since the cytochrome c molecule is a highly chrome c molecule could fit into the crystalline lattice symmetry related peroxidase molecules and interact with the ordered structure, the dynamic disorder is most likely due to peroxidase according to the hypothetical model although some thermal motion of the entire protein and not justindividual adjustments would be required in the positioning of the cy- cytochrome c atoms. Thus, a plausible explanation for why tochrome in order to avoid unfavorably close contacts. Such the cytochrome c molecules do not appear in the electron interactions would not be possiblewith thesecond peroxidase density maps is that the cytochrome c molecules undergo a

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Peroxidase-Cytochrome c Crystals

whole protein thermal vibration relative to the peroxidase such that the average position of each atom of a cytochrome c molecvle exhibits a mean vibration amplitude on the order of 2-3 A. Such motion is sufficiently small that the overall electrostaticinteractions between the two redox partners could be maintained in the crystal in addition to the approximate parallel alignment of the two hemes as predicted by the hypothetical model. Acknowledgments-We wish to thank James Wood and Dave Stewart for performing the electrophoresis experiments. REFERENCES Bechtold, R., and Bosshard, H. R. (1984) J. Biol. Chem. 260, 51915200 Bernstein, F. C., Koetzle, T. F., Williams, G. J . B., Meyer, E. E., and Tasumi, M. (1977) J. Mol. Biol. 112,535-542 Bisson, R., and Capaldi, R. A. (1981) J. Bwl. Chem. 256,4362-4367 Cheung, E., Taylor, K., Kornblatt, J. A., English, A. M., McClendon, G., and Miller, J. R. (1986) Proc. Natl.Acad.Sci. U. S. A . 8 3 , 1330-1333 Crowther, R. A. (1972) in The Molecular Replacement Method (Rossmann, M. G., ed) International Science Review Series, No. 13, Gordon Breach, New York Crowther, R. A., and Blow, D. M. (1967) Acta Crystallogr. 23, 544548 Erman, J. E., and Vitello, L. B. (1980) J. Biol. Chem. 2 5 5 , 62246227 Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1979) in

The Porphyrins (Dolphin, D., ed) Vol. 7, pp. 149-240, Academic Press, Orlando, FL Finzel, B. C., Poulos, T. L., and Kraut, J. (1984) J. Biol. Chem. 2 5 9 , 13027-13036 Fitzgerald, R. M. D. (1987) J.Appl. Crystallogr. 20, in press Gilliland, G. L., Howard, A. J., Winborne, E. L., Poulos, T. L., Stewart, D.B., and Durham, D.R. (1987) J. Biol. Chem. 2 6 2 , 4280-4283 Hendrickson, W. A. (1985) Methods Enzymol. 115, 252-270 Hendrickson, W. A., and Konnert, J. H. (1980) in Computingin Crystallography (Diamond, R., ed) pp. 13.01-13.23, Indian Institute of Science, Bangalore, India Lattman, E. E., and Love,W. E. (1972) ActaCrystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 26, 1854-1857 Liang, N., Pielak, G., Mauk, A. G., Smith, M., and Hoffman, B. M. (1987) Proc. Natl. Acad. Sci. U. S. A . 84, 1249-1252 Maraquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141,369-391 Matthews, B. W. (1968) J. Mol. Biol. 33,491-497 Nelson, C. E., Sitzman, E. V., Kang, C. H., and Margoliash, E. (1977) Anal. Biochem. 83,622-631 Poulos, T. L., and Finzel, B. C. (1984) in Peptide and Protein Reviews (Hearn, M. T. W., ed) pp. 115-171, Marcel Dekker, New York Salemme, F. R. (1977) Annu. Rev. Biochem. 46, 299-329 Simondsen, R. P., Weber, P. C., Salemme, F. R., and Tollin, G. (1982) Biochemistry 21,6366-6375 Takano, T., and Dickerson, R. E. (1981) J. Mol. Biot. 153,95-114 Waldemeyer, B., and Bosshard, H.R. (1985) J. Biol. Chem. 2 6 0 , 5184-5190 Waldemeyer, B., Bechtold, R., Bosshard, H. R., and Poulos, T. L. (1982) J. Biol. Chem. 257,6073-6076