J. Mol. Biol. (1979) 131, 259-285

Structure of a Bacteriochlorophyll a-Protein from the Green Photosynthetic Bacterium Prosthecochloris aestuarii B. W. MATTHEWS, R.E. FENNAt, M.C. BOLOGNESI$, M.F. SCHMID Institute of &?olecular Biology and Department of Physics University of Oregon, Eu,gene, Ore. 97403, U.S.A. AND

J.M. OLSON Biology

Departm,ent, Brookhaven National Upton, N.Y. 11973, U.S.A.

Laboratory

(Received 9 November 1978) The three-dimensional structure of a water-soluble bacteriochlorophyll a-containing protein from the green photosynthetic bacterium Prosthecochloris aestuurii lras been determined by X-ray crystallography from a 2.8 A resolution electron density map based on four isomorphous derivatives. Details of the crystallographic procedures used to obtain the map are presented. The bacteriochlorophyll a-protein is shown to consist of tllror: iderltical subunits, tight,ly packed around a s-fold symmetry axis. Each subunit consists of a cnre of seven bacteriochlorophyll a molecules enclosed within a “bag” of protein. The polypeptide chain forms an extensive l&strand /%sheet, which is almost planar in its central region, and twisted at its extremities, and wraps around the chlorophyll core to form an efficient amphipathic layer between the chlorophylls and the aqueorls environment. There are extensive contacts between the phytyl chains of the seven bacteriochlorophylls within each subunit. These hydrocarbon chains constitute an inner hydrophobic core of the molecule which may be important in forming the complex. There are also extensive contacts between the protein and both the bacteriochlorophyll head groups and tails, but relatively few contacts between the respective head groups. The seven magnesiums all appear to be five co-ordinated. In five cases the presumed ligand is a histidine side-chain, in one case a polypeptide carbonyl oxygen, and in the other case a water molecule. At, low temperature, both the absorption and circular dichroism spectra of the bacteriochlorophyll a-protein show splitting which can be interpreted in general t,erms as due to exciton interactions between the seven chromophores, but calculations of the expected splitting based on the bacteriochlorophyll co-ordinates determined crystallographically are in poor agreement with the observed spectra. F’urthermore, the observed red shift of the QY absorption band of bacteriochloropllyll a, from about 770 nm in organic solvents to 809 nm in the bacteriochloroplryll u-protein, is not explained by the excitor1 calculations. It seems likely that, t,tle red shift is due to perturbations of the spectra of the individual bacteriochlorophylls by the protein environment, but, pending the determination of the amino acid sequence, it is not possible at this time to define in detail all the protein-ol~lorophyll irlterwctions. It, is suggested that the bacteriochlorophyll a-protein t I’rctsent address: Department of Biochemistry, University of Miami School of Medicine, Miami, E’lor. 33152, U.S.A. : l’ermanont address: Istituto de Crititallografia Universitit, via Bassi 4.27100, Pavia, Italy. 259

0022-2836/79/1s0259-27 $02.00/O

$J 1979 Academic

Press Inc. (London)

Ltd.

1~. W.

260

JIATTHEWS

E;

BL.

serves as a good model for the organization of chlorophyll il, ~,i~j, and that t tit* types of interaction seen here between chlorophyll and protein arc’ likely to b*s found in other chlorophyll proteins.

1. Introduction .In the green photosynthetic bacteria, a water-soluble bacteriochlorophyll n-protein is believed to function as an intermediary in the transfer of excit,ation energy from the light-harvesting Chlorobium chlorophyll (Bchlf c. ~1,e or f) t)o the photochemical reaction centers (Olson & Romano, 1962 ; 8ybesma & Olson, 1963: SSbesma & Vredenberg, 1963,1964). We describe here the three-dimensional xtrurture of the Bchl u-protein from Prosthecochloris nestuarii, strain 2K, as determined by S-rab crystallography from a 2.8 A resolution electron densit>y map. (The skain of bacteria was previously identified incorrect>ly as ChloropseufEomorans ethylicn or Chlorobium Zimicola (Olson, 197&x).) Preliminary crgstallographio results (Forms et nb.. 1974) showed that the molecule is a trimer of molecular wright 150,000 and t#hat eac.11of the three identical subunits contains seven molecules of Bchl n (Fenna $Mahthews. 1975,1976). The initial electron densit,y map was based on bhree inomorphons heavy-at,om derivatives. In this paper we describe the X-ray crystallographic procedures usrtl to obtain a better electron density map, improved by the inclusion of a fourth derivative and by remeasurement of some of the data. A more detailed analysis of the structure is presented, and an attempt is made to rationalize the optical properties of the Bchl a-protein in the light of the t,hree-dimensional structure.

2. Crystallization Bacteriochlorophyll

a-protein was prepared from mixed cultures of P. aestuarii wntl Crystals suit,able for X-ray diffract,ion Desulfuromonas acetoxidans (Olson. 1971.197%). analysis were obtained by a method based on that of J. M. Olson et al. (I 969). Two droplets, one containing protein and the other precipitant were placed about 1 cm apart on a plast)ir sealed in a Petri dish and or glass plate, joined by a narrow V-shaped liquid junction, allowed to equilibrate, presumably by a combination of vapor diffusion a,nd salt diffusion via the junction. The protein droplet contained 50 ~1 of the Bchl prot,ein. -14 mg ml- I. in 10 mM-Tris*HCl buffer (pH 7.8), 1 M-NaCl, the precipitant droplet 50 ~1 of 10~~~ (\v,‘v) ammonium sulfate in the same buffer/NaCl mixture. The crystallization dishes were kept at 5°C and the crystals grew as hexagonal rods up to 0.5 mm diam. and 1 mm lotkp. OYF’I several weeks. In order to minimize excessive nucleation. it, was found helpful to centrifuge the protein solution twice for at least 40 min at 27,000 g, carefully transferring t,he supernatant between runs, before setting up the crystallization experiments. Prior t)o X-ra? photography, and for storage, the crystals were equilibrated wit,h a “standard mot,hrl liquor”, the same as that in the precipitant droplet described above. 98~4 A. The crystals have space group P6, with cell dimensions a =- h _-- 1124 ‘4, c: with 1 subunit/asymmetric unit, implying that the Bchl protein ronsistn of 3 identical subunits arranged with S-fold symmetry (Fenna et al., 1974). The packing in t,ho crystals was very open, with only about 30% of the crystal occupied by Bchl protein, and tllo remainder by solvent, and was characterized by large channels, about 62 A in diameter, parallel to the hexagonal axis, extending through the crystals. These channels can be readily seen in electron micrographs of stained crystals (J. M. Olson et al., 1969; R. .4. Olson et ul.. 1969; Labaw & Olson, 1970). The X-ray structure determination has confirmed t’he overall molecular packing of the Bchl protein proposed from the electron microscope analysts (Matthews et al., 1977), although the molecules arc now known tlb be t,rirners rather t#hatt tetramers, as assumed in the earlier studies. t Abbreviations

used: Bchl, bacteriochlorophyll.

BACTERIOCHLOROPHYLL

n-PROTEIN

261

3. Data Collection and Processing Diffraction data from the native crystals and heavy-atom derivatives were recorded photographically on Kodak no-screen X-ray film using an Elliott GX6 rotating anode X-ray generator operating at 1600 W and Enraf-Nonius precession cameras. A 0.4 mm standard collimator was used and the copper K/I radiation reduced by means of a nickel foil filter. The crystal-to-film distance was 75 mm. The exposure time for each photograph was about 40 hours, dependent upon the size of the cry&al, which was normally 0.45 mm in the shortest dimension. A complete three-dimensional data set to 2-8 A resolution required 23 screened precession photographs including the reciprocal lattice planes: hkn (YL= 0,3); hnl (n = 0,8); h(h--n)E (n = 0,9). This scheme, illustrated in E’igure 1, includes 90% of

2.0 a L,k , (h,n.0

FIG. I. Data collection schrm~. the reflections to 2.8 A, 43% between 2.8 A and 2.7 A, and allows ample overlap between levels for scaling purposes. The direction of po&ive 1 in the reciprocal latt,ice was chosen arbitrarily and then individual films of the types hnl or h(h-,n,)l were indexed in a manner consistent wit,h this choice. For these planes, the Friedel pairs hkl and hki can be measured from the same film. One-half of each film includes refiections indexed h,n,fZ or h,(h-n),+E while the other half includes those with indices n.k;&l or k,(k-n),fZ. Depending upon the orientation of the crystal with respect to the direction of the X-ray beam, photographs of the type hkn have reflections with either positive Z or negative 1 for the whole film, so that no anomalous scattering differences are obtained from these films. The choice of the sign of Z is readily made; for example by inspection of the hk0 zone on a setting photograph.

162

B. W. MATTHEWS

lC;IT A.L.

Film optical densities at points on a two-dimensional grid of raster size 100 ,LL~I were measured with an Optronics rotating drum microdensitometer operated on line to a Varian 620/i computer (Matthews et al., 1972). Individual intensities wert~ Oaken as the sums of O.D. measurements within a rectangular box (typically 11 x !4 raster points) centered at the refined reciprocal lattice points. Background measurements were made vertically above and below reflections at points centered midway between reciprocal lattice rows. The number of point background measurements generally exceeded the number of point measurements for t,he reflections. d pair of films was used for each exposure, and comparison of front and back films enabled an estimate of the film non-linearity correction factor t*o btb calculated for faaoll exposure (Matthews et al., 1972). Measurements which included point density readings exceeding 2.5 O.D. units were discarded. Before applying appropriate Lorentz and polarization corrections; t#he intensitic,s of symmetry-related reflect’ions recorded from hkn, h0Z and hhb films were averaged. Symmetry H factors were computed for these measurements together w&h the Friedel-related pairs of reflections on films of the types haul or h(h--n)Z with u :a 1. Each isomorphous derivative data film was scaled directly to the corresponding native film for each of t,he 23 different reciprocal lattice planes in t~hethree-dimensional data set. In an attempt to maximize the quality of the data: poor films. as judged by the values of the R factors for symmetry and scaling, were discarded and repeat measurements made on new crystals. Tn a few cases where duplicate films of similar quality had been obtained these were averaged before inclusion in the data srt . In all. duplicates for three native, five platinum, two mercury and four many1 lattice plant~~ were included. TAocal scaling to reduce systematic errors in the measurement of Friedel differences was tested according to the criteria described by Matthews KCzerwinski (1975). This procedure was found to produce significant improvement in 20% of the derivative films and was applied only t’o these films. Scale factors between films comprising the native three-dimensional data set, were computed by the method of Hamilton et al. (1965). The same scale factors were then applied to each of thtl individually scaled derivative data films. Typically each set of 23 precession photographs gave 78,000 intensity measurements, which reduced to about 17.000 unique amplitudes after symmetry averaging and merging. In determining t,he scale fact-orb between planes, t)he weaker amplitudes corresponding to 25%, of the t,ot,al of 5,50() overlaps were omitted. The merging R factors quoted in Table I are, howcavcr. calculated from all overlapping measurements. CorreIat,ion coefficients Cij -= X4 ,A,/(L’A~ZLIS)* b c,at‘ween bot’h isomorphous antI anomalous int,ensity differences recorded on more Ohan one precession phot,ograph were computed as described by Colman et al. (1972) and Remington rt d. (1978). This procedure served as an independent check on the self consistency of indexing within each dat,a set,. The overall correlation coefficients are 1ist)ed in Table 1 t~ogcthrr with the other statistics compiled during the data processing.

4. Heavy-atom Derivatives and Phasing Four isomorphous heavy-atom derivatives were used in the Bchl protein structure analysis. ;\ platinum derivative was made by soaking nat,ive crystals for seven days in 0.5 mlvr-potassium chloroplatinite in standard mother liquor consisting of 10’;,, (w/v) ammonium sulfate, 1 M-sodium chloride and 0.01 M-Tris.HCl (pH 7.8). The

BACTERIOCHLOROPHYLL

263

n-PROTEIN

TABLE

1

Data processing statistics Tjata

set

Unique reflections Average R,,,(l)t ;Iverage R,,,(2) .iverage R,,,, R merge Overall isomorphous rorrelation$ Overall anomalous correlat,ion

Native

K,PtCl,

MeHgT

Pt -1 Hg

KdUW,F,

16,608 0.058 0.107 0.072 0.046

16,995 0.063 0.100 0.082 0.051

17,004 0.062 0.103 0.072 0.047

16,948 0.058 0.099 0.064 0.049

16,746 0.069 0.101 0.064 0.043

-

0~38

0.68

0.71

0.79

-

0.02

0.05

0.08

0.34

t The R values are as defined previously (Matthews et nl., 1972). R,,, gives the agreement planes refers to hnl and h(h--n)Z between symmetry-related reflections on the same film. R ,,,(l) having 2 symmetry-related reflections or 4 for h0Z and hhl. R ,,,(2) refers to the hkn planes having R ScSl gives the agreement between symmetry-averaged intensities 6 symmetry-related reflections. recorded on the stronger and weaker film in a film pack. R merge gives the agreement between struct,ure amplitudes measured on different films. 1 The correlation coefficients are overall values as described in the text.

second, mercury, derivative was obt,ained by soaking the crystals for seven days in a saturated solution of methyl mercuric iodide in the same mother liquor. A third. double derivative, was made by soaking crystals for seven days in the presence of both O-5 mM-potassium chloroplatinite and saturated methyl mercuric iodide. The fourth derivative was formed by soaking crystals for two days in 20 mM-K,(UO,),F, in 10% (w/v) ammonium sulfate and 1 M-sodium chloride buffered with 0.05 MPIPES at pH 6.5. In order to avoid crystal cracking, the reduction in pH from 7.8 to 6.5 was carried out in steps of 0.2 of a pH unit, accompanied by a change in buffer from Tris t.o PIPES at pH 7.0. Comparison of the diffraction patterns recorded from native crystals at pH 7.8 and pH 6.5 showed no significant changes in intensities or in the unit cell dimensions. Single sites in each of the platinum and mercury derivatives were initially located from dieerence Patterson syntheses of the centro-symmetric hk0 zone using data t#o 5 A resolution. In space group P6,, self vectors occur at (z,y), (22,2y) and (211:- y. z -+ ,y). In the platinum derivative difference Patterson (Fig. 2(a)) one of the double weight vectors occurs as the highest peak in the map. In the mercury derivative difference Patterson (Fig. 2(b)) a possible first site appeared as a set of three superimposing vectors, and hence occurring with multiple weight. This entirely fortuitous superposition of the three self vectors greatly facilitated the location of the first, mercury site in this derivative. Two further mercury sites were found in a difference Fourier synthesis of the hk0 zone using phases calculated from the platinum and first mercury sites. Subsequently, the relative z co-ordinates of the platinum and mercury sites were determined from three-dimensional difference Patterson and Fourier syntheses. Similarly, sites in the platinum plus mercury double derivative and the uranyl derivative were first located in projection difference Fourier syntheses and the z co-ordinates later determined from three-dimensional Fourier syntheses phased on the platinum and mercury single derivatives.

FIG. 2. Difference Patterson projections at 5 A resolution for the hk0 zone. Self vectors ( x ) and (+), cross vectors (z#); symbols for double-weight peaks are drawn heavily. (a) Platinum derivative. (b) Mercury derivative showing two major sites.

The heavy-atom parameters for all four derivatives were then refined by using a lack-of-closure least-squares refinement procedure applied to a subset of the t)hreedimensional data, selected to include all the centric data together with 25o/0 of the acentric reflections. Choice of the correct enantiomorph for the heavy-abom distribution was made by including phasing contributions from tjhe anomalous components of the heavy-atom scattering in two separate refinement cycles, in one of which the heavy-atom co-ordinates were the mirror image of those used in the other cycle (cf. Matthews, 1966,197O). One of the choices was found t’o give significantly E’, for the lower values (4 to 10%) for the root-mean-square lack of isomorphism, anomalous contributions of each of the derivatives. The low values found for the correlation of anomalous differences measured on different films (Table 2) indicate that, for the platinum derivative in particular, these differences are barely significant’. and, for this reason, the anomalous contributions of the K&Cl, derivative were not, included in the final phase calculation (North, 1965; Matthews, 1966). The heavy“best” phase calculation (Blow & Crick. atom parameters used in the four-derivative 1959) are listed in Table 2. The program for the refinement was developed by Dr 1,. F. Ten Eyck and that for the Fourier synthesis by G. N. Reeke. A summary of the heavyatom refinement statistics is given in Table 3 and the ratio of the root-mean-square heavy-atom scattering to the lack of closure, E, for each derivative and the figure of merit for the phasing are given as a function of the resolution in Figure 3. The overall mean figure of merit was 0.74 and the average ratio of the lack of closure of the phaso triangles to E was 0.93 (Matthews, 1970). Normally, three isomorphous heavy-atom derivatives might be considered sufficient, for reasonably accurate phase determination, but this was not the case for the Bchl a-protein for the following reasons. (1) The introduction of a single platinum atom into an asymmetSric unit of 45,000 molecular weight produced only small changes in the diffracted structure amplitudes (about 10%). Considerable efforts were made to minimize errors in the intensity measurement, and the correlation coefficient of 0.38 (Table 1) indicates that the platinum isomorphous differences are significant, but this derivative nevertheless contributes weakly to the overall phase determination”

BACTERIOCHLOROPHPLT~

rs-Z’ROTEIS

TABLE

“65

2

Heavy-atom parameters

(1 site)

27.0

0.113

0.443

~~ 0.251

38.1

(3 sites)

38.5 15.6 8.2

0.380 a.301 0.278

0~583 0.439 0.434

0.007 --0.103 --0.141

26.3 40.0 10.7

32.2 37.4 13.5 8.5 8.6

0.113 0.380 0.301 0.278 0.511

0.443 0.582 0.438 0.434 0.204

-0.25 0.007 -0.104 -0.142 - 0.396

43.3 27.8 36.5 12.7 29.6

34.5 19.1

0.437 0.504 0.324

0.502 0.495 0.022 0.512 0.415 0~050 0.292 0.327 0.462 0.655 0.370

K,Pt,Cl, MPH@

K,PtCI,

+ MeHgI

I