Association For Molecular Pathology et al v. United States Patent and Trademark Office et al

Filing 170

DECLARATION of JOSEPH SCHLESSINGER. PH.D. in Support re: 61 MOTION for Summary Judgment., 152 MOTION for Summary Judgment.. Document filed by Myriad Genetics, Lorris Betz, Roger Boyer, Jack Brittain, Arnold B. Combe, Raymond Gesteland, James U. Jensen, John Kendall Morris, Thomas Parks, David W. Pershing, Michael K. Young. (Attachments: # 1 Exhibit 1, # 2 Exhibit 2 Part 1, # 3 Exhibit 2 Part 2)(Poissant, Brian)

D

ockets.Justia.com

1

His 378
*

CUB

Table 2. Collection of data and statistics of phase determination. The crystals diffracted x-rays up to 2.6 A resolution. The crystal of the enzyme belongs to the orthorhombic space group of P212121 with unit cell dimensions a = 189.1 A, b = 210.5 A, and c = 178.6 A, containing two molecules in an asymmetric unit. Intensity data were collected with monochromatized x-ray of 1.0 A at the Photon Factory, Tsukuba, Japan, by means of a modified Weissenberg camera for macromolecules (24). Diffraction intensities were processed with the program DENZO (25). Searching more than 50 heavy atom derivatives with MAC Science imaging plate detector (DIP2000) on a Rigaku rotating anode x-ray generator (RU300), we yielded three derivative data sets available for phase determination. Two of three derivatives were prepared by soaking the crystals in Na2lrCl6 solutions at various concentrations. The third was a CH3HgCI derivative. Heavy atom sites of each derivative were located by solving each difference Patterson function at 5 A resolution. The heavy atom parameters of three derivatives were refined with the program MLPHARE (26) of the CCP4 program package. In that MIR (27) phase information was poor beyond 5 A resolution, phases were extended from 5 to 2.8 A in 200 small steps with the use of solvent flattening (28) by means of the program DM of CCP4. The protein mask during the phase extension was determined by the Wang method (28) with a sphere of 10 A and 70 percent solvent content by volume. A free R factor (29) for 5 percent of the reflections in each shell was reduced from 0.53 to 0.28. The noncrystallographic symmetry relation between two molecules in an asymmetric unit was determined, and the correlation coefficient {((pl - (p1))(p2 - (p2))/Y;[(pl - (p1))2(p2 - (p2))2]1/2} was as high as 0.78. The phase refinement procedure including NCS averaging (30) was undertaken, starting at 5 to 2.8 A resolution. The free R factor and the correlation coefficient at the final stage were 0.26 and 0.92, respectively.
Native

heme a A

heme

Phe 377

heme a3

u

Fig. 3. Structural relation between two hemes in the active site of cytochrome c oxidase. Red structures and green structures represent hemes and amino acid residues in subunit 1. A blue ball and red balls define the positions of copper and iron atoms, respectively. The hemes a and a3 are bridged by three successive residues, His376, Phe377, and His375 in the helix X.

lrCl6(l)
100-3.0 (3.2-3.0) 319,358 (18,599) 125,036 (11,545) 10.0 (1.4) 2.6

IrCI6(ll)
100-3.0 (3.1-3.0) 381,828 (28,410) 133,245 (14,008) 21.3 (2.7) 2.9

CH3HgCI
100-3.0 (3.1-3.0) 328,484 (15,186)

Resolution ranges (A)* Observed reflections

Independent reflections

Ihrf(I)
Averaged redundancyt

100-2.8 (2.9-2.8) 518,054 (24,542) 159,742 (13,074) 19.5 (2.2) 3.2

127,548
(9,396)
2.4

His 240

(1.9) Completeness (t)
90.1

(1.6)
86.3

(2.0)
93.0

(1.5) 2.6 (1.6)
88.3

Rmerge§
Risoll

(74.4) 7.8 (29.0)

(64.5) 9.0 (29.6)
7.3

(79.1)
6.2

(65.7)
8.5

(23.4)
5.7

(28.4)
9.0 (11.4) 0.79 (0.95)

Rculfis91
Phasing power#

(11.6) 0.90 (0.97) 0.38 (0.18)

(8.7) 0.86 (0.97) 0.54

0.74

(0.30)

(0.34)

tRedundancy is the number of observed *Figures in parentheses are given for the highest resolution shells. reflections for each independent reflection. tRmerge, Yh, i(hi) - ('(h)) Iyhl/(h,i), where /(h,i) is the intensity value of the i-th measurement of h and (1(h)) is the corresponding mean value of 1(h) for all i measurements; the summation is ea over the reflections with 1/u(I) larger than 1 .0. IlRiso, E FPH-I FP /XIFP where FPH and FP are theI,dwrhievrteive and thies FH(calc) native structure factor amplitudes, respectively. śR0c11,i, E FPH - Fp| - FH(calc) /E FPH FP #Phasing power is the calculated heavy atom structure factor. The summation is over the centric reflections only. root-mean-square (rms) isomorphous difference divided by rms residual lack of closure.

Fig. 4. A side view of the 02 binding and reduction site. The red structure is heme a3 and the green ones are amino acids. The dotted line shows a possible hydrogen bond between the hydroxyl of the hydroxyfarnesylethyl group and Tyr244. The broken lines represent coordination bonds. The red and green balls represent the positions of iron and copper atoms, respectively. A close contact between Tyr244 and His240 with both aromatic rings oriented perpendicular with each other is also shown.

1072

SCIENCE

*

VOL. 269 * 25 AUGUST 1995

Downloaded from www.sciencemag.org on December 20, 2009

ed structure is applicable for the crystal structure. Helix VII is two residues shorter and is shifted 10 residues toward the NH2terminal, compared to the helix predicted. A zinc atom was close to the molecular surface in a nuclear-coded subunit Vb on the matrix side. The magnesium site was near CuA at the interface of subunits I and II. The electron transfer path within the enzyme has essentially been established recently after long and extensive efforts as follows: cytochrome c -> CuA-> heme a -> the 02 binding sites, which includes heme a3 and CUB (13). The location of CuA seems consistent with its role as the initial electron acceptor from cytochrome c. However, this overall location of the metal site does not exclude the possibility for a direct electron transfer from CuA to the heme a3-CuB site. Structures of two hemes and CUB sites. Both heme planes were essentially perpen-

dicular to the membrane plane facing each other at an angle of 1040. Heme a was coordinated with two imidazoles of histidine residues (His61 in helix II and His378 in helix X). The fifth ligand of heme a3 was an imidazole of His376 in helix X, whereas CUB was coordinated by three imidazoles of His240 in helix VI and His290 and His291, both in a nonhelical fragment between helices VII and VIII. These ligations are consistent with those proposed as a result of mutagenesis experiments (12). The heme a3 plane was positioned so that the pyrrole ring with a hydroxyfarnesylethyl side chain and the one with a vinyl side group were placed at lower right and left sides, respectively, when viewed from the CuB side, with the cytosolic side being considered as the upside. Thus, the pyrrole with methyl and propionic acid groups was located at the upper left side and the one with a formyl

and propionic acid groups was located at the upper right. In the case of heme a, the pyrrole with a hydroxyfarnesylethyl side chain was at the lower left, viewed from the CUB side (Fig. 2). The hydroxyfarnesylethyl group of heme a was almost in the extended conformation, held by helices 1, 11, X, XI, and XII, and that of heme a3 was twisted to

His 376

%K4*A-alwoomm

S

.l

AS

form a U-shaped arm, located between helices VIII and IX. None of the hydroxyfamesylethyl side chain interacted with the redox centers or the porphyrin aromatic ring system, contrary to the proposals based on the kinetic behavior of the enzyme (4, 14). A phenyl plane of a phenylalanine (Phe377 in helix X) was observed halfway between the two planes of the heme a3 and one of the imidazole ligands of heme a (His378) (Fig. 3). The distances between the phenyl plane and the two planes on both sides are as short as 3.5 A. Thus, any change in the orientation and position of the phenyl group, induced by change in the ligation

Fig. 5. A representation of CUA site showing a structure. Green balls show copper atom positions. Blue broken lines indicate coordination bonds. The peptide carbonyl group of Glu198 is illustrated with a bar marked with C and 0.

[2Cu-2Sy]

A

B
CuA C
*

~~~~M

g

GIu 198 H20
.004

heme a

heme a3
His 368

Fig. 6. The magnesium site and its relative orientation to other metal centers. (A) Coordination of magnesium. Green and light gray colors denote amino acid residues of subunits and Ill, respectively. Dark orange and blue balls are at the positions of magnesium atom and oxygen atom of water, respectively. Blue broken lines are coordination bonds forming a distorted tetrahedron. (B) Location of the magnesium site and other active centers. Belonging of amino acids is shown with the same colors as in (A). Positions of iron, copper, magnesium, and oxygen of water are indicated with red, blue, dark orange, and dark blue balls, respectively.
SCIENCE * VOL. 269 * 25 AUGUST 1995

1 073

Downloaded from www.sciencemag.org on December 20, 2009

and oxidation state of Feda from the fifth ligand imidazole of His376, adjacent to Phe377, could control the electron transfer between Fea and Fea,3 A side view of the heme a3-CuB structure reveals the phenol OH group of tyrosine, Tyr244, in subunit I oriented to the OH of the hydroxyfamesylethyl group (Fig. 4). The CUB site was coordinated only by the three imidazoles of His240, His290, and His291. No other ligand to CUB, even water, was detected. The high spin Fe,3 ion was slightly displaced from the heme plane toward the fifth ligand. The CUB was 4.5 A away from Fea3 and 1.0 A from the heme normal at Fea3 position toward the nitrogen atom of the pyrrole with the formyl group. One of the unexpected findings was the absence of a ligand directly bridging the Fea3 and CUB. A bridging ligand has been proposed on the basis of strong antiferromagnetic coupling between the two atoms, a characteristic that was discovered 26 years ago (15); other model structures with a bridging ligand have been proposed (16). The presence of an amino acid side chain randomly oriented so that the electron density could not be detected by x-ray crystallographic analysis is unlikely because all the amino acid side chains near the dinuclear site have been assigned. However, the presence of water molecules nonspecifically and randomly trapped cannot be excluded. A typical hydrogen bond is likely to be present between the hydroxyl groups of a tyrosine (Tyr244) and the hydroxyfamesylethyl side chain of heme a3 in that the

oxygen-oxygen distance is 3.0 A. Furthermore, the orientation of the phenol plane of Tyr244 was perpendicular to the imidazole plane of His240, which liganded to CUB. The nitrogen atom of the imidazole of His240 was located 3.1 A above the plane of Tyr244. The system, including the imidazole phenolhydroxyfarnesylethyl side chain, could facilitate an electron transfer pathway from Fea to CUB. A structure that could provide another electron transfer pathway from heme a3 to CUB is the formyl side group at the pyrrole, located 3.0 A above the imidazole plane of His290, which is a ligand of CUB. Although the distance between Fea and CUB, as short as 4.5 A, suggests a facile electron transfer directly between the two metals, multiple electron transfer pathways are still possible for dioxygen reduction. In contrast, these structures may also induce an oxidation state-dependent conformational change to couple 02 reduction with proton pumping. When Tyr244, which is not bound directly to CUB, was replaced by phenylalanine, CUB was disrupted (12), suggesting some structural role of the amino acid. Other interesting structures near the heme a3 and CUB site are a hydrogen bond between the imidazole of His376 (the fifth ligand of heme a3) and the peptide carbonyl of Gly351, and a parallel nT-I contact between His291 of CUB ligand and Trp236. The imidazole of a heme a ligand, His61, is linked to a carbonyl oxygen of Gly30 with a hydrogen bond. No close contact with the formyl group of heme a is detectable. Structure of CUA. The CuA site in subunit II consists of six ligands, two cysteines (Cys'96 and Cys200), two histidines (His'16 and His204), a methionine (Met207), and a peptide carbonyl of a glutamate (Glu198) (Fig. 5). All these ligands, except for the peptide carbonyl, have been proposed as a result of mutagenesis experiments (17). The anomalous Fourier map at the central metal site exhibited a single peak (Fig. 1), which suggested a single metal, although both the peak heights in the Fourier and the anomalous Fourier maps, respectively, were too high to be attributable to a single copper atom (Table 1). The arrangement of these six ligands, determined from the electron density distribution near this site, does not provide any reasonable position for a single copper atom, but is fully consistent with a dinuclear copper center, as follows. Two copper atoms are bridged by two sulfur atoms of Cys'96 and Cys200, placing the four atoms on the same plane with the interatomic distances of 2.7 A (Cu-Cu), and 3.8 A (S-y-Sy). One of the copper atoms is coordinated by the imidazole nitrogen of His'6' and the methionine sulfur of Met207, forming a tetrahedral coordination including the two cysteine sulfur atoms. Coordination of the other copper is also tetrahe-

now.

offiplum

Nwom

lnniolowm~

dral, including the imidazole nitrogen (His204) and the peptide carbonyl (Glu'98). The geometry is similar to that of a [2Fe2S]-type iron-sulfur center (18), in which the Fe ions and inorganic sulfur atoms are replaced with Cu ions and cysteine sulfur atoms, respectively. Both dinuclear centers stabilize a one-electron delocalized oxidation state, that is, [Fe25+---Fe25+] in the reduced form of [2Fe-2S] center and [Cul'5+---Cu'.5+] in the oxidized state of [2Cu-2Sfy] center of CuA site. Recently, a dinuclear Cu center for CuA has been proposed in analogy to that of the copper enzyme N20 reductase for which various structures not fully consistent with our structure have been suggested (19). Structures of zinc and magnesium sites. Tetrahedral coordination was detectable for the zinc site where four cysteines (Cys60, Cys62, Cys82, and Cys85) in subunit Vb are coordinated by the central atom (Fig. 1C). The polypeptide fragment from Cys60 to Cys82 shows a zinc finger motif. However, the fragment does not form any protrusion from a globular core of the protein subunit, contrary to the typical zinc finger structure which interacts with a double-stranded DNA (20). The electron density distribution of the enzyme crystals showed several other coordination structures, each with a central atom, that did not show any anomalous scattering for the x-rays at 1.0 A. One of them involved an aspartate (Asp369) and His368 in subunit I, Glu198 in subunit II, and an oxygen atom of water as the ligands in a distorted tetrahedral coordination (Fig. 6A). In agreement, Ferguson-Miller et al. discovered that His411 and Asp412 of Rhodobacter sphaeroides, which corresponds to His368 and Asp369 in beef heart enzyme, respectively, are essential for manganese binding to the enzyme at a site for which magnesium competes (21). Furthermore, electron density at this site is at a level reasonable for a magnesium atom. Thus, this site is highly likely to be a magnesium site. The magnesium site was found near (approximately 6 A away from) two adjacent arginine residues (Arg438 and Arg439)

REFERENCES AND NOTES
1 R. A. Capaldi, Annu. Rev. Biochem. 59, 569 (1990). 2. B. G. Malmstrom, Chem. Rev. 90,1247 (1990). 3. G. T. Babcock and M. Wikstrom, Nature 356, 301

(1992).
4. 0. Einersdottir et al., Biochemistry 32, 12013 (1993). 5. W. S. Caughey, A. Dong, V. Sampath, S. Yoshikawa, X. -J. Zhao, J. Bioenerg. Biomembr. 25, 81 (1993). 6. We follow the terminology of Kadenbach for the subunits of cytochrome oxidase [B. Kadenbach and P. Merle, FEBS Lett. 135, 1 (1981); B. Kadenbach, U. Ungibaver, J. Jaraush, U. BOge, L. Kuhn-Neutwig, Trends Biochem. Sci. 8, 398 (1983)). 7. 0. Warburg and E. Negelein, Biochem. Z. 214, 64

(1929).
8. S. Yoshikawa, T. Tera, Y. Takahashi, T. Tsukihara, W. S. Caughey, Proc. Natl. Acad. Sci. U.S.A. 85, 1354 (1988). 9. S. Yoshikawa et al., in preparation.

20. T. Hard et al., Science 249, 157 (1990). 21. J. P. Hosler, M. P. Espe, Y. Zhen, G. T. Babcock, S. Ferguson-Miller, Biochemistry 34, 7586 (1995); M. P. Espe, J. P. Hosler, S. Ferguson-Miller, G. T. Babcock, J. McCracken, ibid., p. 7593. 22. 0. Einersdottir and W. S. Caughey, Biochim. Biophys. Res. Commun. 129, 840 (1985); G. C. M. Steffens, T. Souliname, G. Wolf, G. Buse, Eur. J. Biochem. 213, 1149 (1993); M. Oblad et al., Biochim. Biophys. Acta 975, 267 (1989). 23. R. A. Scott, Annu. Rev. Biophys. Biophys. Chem. 18, 137 (1989). 24. N. Sakabe, J. Appl. Cryst. 16, 542 (1983). 25. Z. Otwinowski, DENZO, "A film processing program for macromolecular crystallography" (Yale University, New Haven, CT, 1985). 26. _ , MLPHARE CCP4 Proc., p. 80 (1991) (Daresbury Laboratory, Warrington, UK). 27. D. W. Green, V. M. Ingram, M. F. Perutz, Proc. R. Soc. London Ser. A 225, 287 (1954). 28. B. C. Wang, Methods Enzymol. 115, 90 (1985). 29. A. T. Brunger, Nature 355, 472 (1992). 30. G. Bricogne, Acta Crystallogr. A 30, 395 (1974); ibid. 32, 832 (1974). 31. T. A. Jones, J. Appl. Cryst. 11, 268 (1978). 32. Supported in part by the Ministry of Education and Culture of Japan Grants-in-Aid for Scientific Research on Priority Area [Bioinorganic Chemistry and Cell Energetics (S.Y.), and 06276102 and 05244102 (T.T.)]; Grants-in-Aid for Scientific Research 06558102 (T.T.). The work was done with the approval of the Photon Factory Advisory Committee, the National Laboratory for High Energy Physics, Japan (proposal 91-050 and 94G-041). We thank N. Sakabe, A. Nakagawa, N. Watanabe, and S. Ikemizu for help in data collection by means of the Weissenberg camera and synchrotron radiation, Y. Morimoto for advise in computation, and S. Ferguson-Miller for the results about Mg binding site in the enzyme before publication. 3 July 1995; accepted 24 July 1995

1074

SCIENCE * VOL. 269 * 25 AUGUST 1995

Downloaded from www.sciencemag.org on December 20, 2009

that faced diagonally to two propionic acid side groups of pyrroles of both hemes to form a tetrahedron involving two positively charged and two negatively charged groups. One of the ligands of the magnesium site, Glu198 of subunit II, was also coordinated to CUA at the peptide carbonyl, suggesting an oxidation state-linked conformational change which controls the electron transfer pathways between CuA and Fea and between Fea and Fea3 via the Arg-propionic acid system as stated above. A structural role of the magnesium for stabilizing the CuA site is also possible in that mutagenesis data suggest that Glu`98 is required for forming the CuA site (17). The magnesium site is located on the hypotenuse of a rightangled triangle including Fea, Fea3, and CUA with Fea3-CuA as the hypotenuse (Fig. 6B). The presence of zinc and magnesium in equimolar amount of heme a3 has been reported from several laboratories (8, 22). However, the roles of these metals have not been clear. Our data clearly indicate the presence of intrinsic zinc and magnesium. The above metal site structures are not completely consistent with the structures obtained from EXAFS. The inconsistency may be due to differences in the model used for the analysis of EXAFS data (23). The active site structures obtained here, although they resolve many controversies, require further refinement for the elucidation of the reaction mechanism of this enzyme.

10. S. Yoshikawa, M. G. Choc, M. C. O'Tool, W. S. Caughey, J. Biol. Chem. 252, 5498 (1977). 11. G. M. Baker, M. Noguchi, G. Palmer, ibid. 262, 595 (1987). 12. J. P. Hosler et al., J. Bioenerg. Biomembr. 25, 121 (1993); B. L. Trumpower and R. B. Gennis, Annu. Rev. Biochem. 63, 675 (1994). 13. B. C. Hill, J. Biol. Chem. 266, 2219 (1991). 14. W. S. Caughey et al., ibid. 250, 7602 (1969). 15. B. F. Van Gelder and H. Beinert, Biochim. Biophys. Acta 189,1 (1969). 16. S. C. Lee and R. H. Holm, J. Am. Chem. Soc. 115, 5833 (1993); A. Nanthakuman et al., ibid., 8513; G. Palmer, G. T. Babcock, L. E. Vickery, Proc. Natl. Acad. Sci. U.S.A. 73, 2206 (1976); C. H. A. Seiter and S. G. Angels, ibid. 77,1806 (1980). 17. M. Kelly et al., J. Biol. Chem. 268,16781 (1993). 18. T. Tsukihara et al., J. Biochem. 90, 1763 (1981). 19. P. M. H. Kroneck, W. A. Antholine, J. Riester, W. G. Zumft, FEBS Lett. 247, 70 (1988); B. G. Malmstrom and R. Aasa, ibid. 325, 49 (1993); N. J. Blackburn, M. E. Barr, W. H. Woodruff, J. van der Oost, S. de Vries, Biochemistry 33, 10401 (1994); H. Bertagnolli and W. Kaim, Angew. Chem. Int. Ed. Engl. 34, 771

(1995).