Stuart, D. S. &Artymiuk, P. (1985) Acta Crystallogr. Sect. A 40,. 713-716. 26. Brunger, A. T., Kuriyan, J. & Karplus, M. (1987) Science 235, 458-460. 27. Connolly ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 6533-6537, September 1989 Biochemistry
Crystallographic structure of a photoreceptor protein at 2.4 A resolution (photocycle/photoactive yellow protein/photoisomerization/sensory rhodopsin/xwray diffraction)
DUNCAN E. MCREE*, JOHN A. TAINER*, TERRY E. MEYERt, JOSEF VAN BEEUMEN*, MICHAEL A. CUSANOVICHt, AND ELIZABETH D. GETZOFF* *Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037; tLaboratory of Microbiology, State University of Ghent, Ghent, Belgium; and tDepartment of Biochemistry, University of Arizona, Tucson, AZ 85721
Communicated by Sydney Brenner, April 17, 1989
lactoglobulin (13, 14). The sequence homology of RBPs and the odorant-binding proteins has led to the suggestion that this /3-fold may play a role in signal transduction by interacting with cellular receptors in a manner dependent upon ligand binding (15). Atomic structures are known for two proteins with photochemistry: bacteriochlorophyll a protein (16), which transmits light energy to the reaction center, and the photosynthetic reaction center (17), which transfers an electron to quinone when excited. PYP differs from these proteins in that it undergoes a photoisomerization when excited; the absorbed photon causes a structural rearrangement that is completely reversible in the absence of other substrates or redox activity. We have undertaken determination of PYP's three-dimensional structure to delineate the molecular events involved in photoactivity in this and other proteins with a photocycle, such as the rhodopsins and bacteriorhodopsin. We describe here the three-dimensional fold and the stereochemical environment of the chromophore in the dark, unbleached form of PYP as determined at 2.4 A resolution by the method of multiple isomorphous replacement (MIR).
The first essential step in protein photorecepABSTRACT tion is the capture and storage of energy from a photon. We have recently identified and isolated, from the purple photoautotrophic bacterium, Ectothiorhodospira halophia, a 13,000dalton photoactive yellow protein (PYP) that has a photocycle with kinetics similar to sensory rhodopsin and a very high quantum efficiency. To study the structural chemistry of protein photoreception, we determined, refined, and analyzed the crystallographic structure of PYP at 2.4 A resolution and report here that it is composed of two perpendicular antiparallel «-sheets that enclose the chromophore. Each of the 10 fl-strands of PYP is connected directly to its nearest neighbor with + 1 topology. Globally, an asymmetric distribution of side chains places aromatic and acidic side chains in an ellipsoidal band around the chromophore with a cluster of basic side chains on one side. Locally, the electron density maps place an internal lysine and the chromophore in an apparent Schiff base linkage stabilized by a buried glutamate and a tyrosine side chain. To our knowledge, the atomic resolution structure of a protein with a reversible photoisomerization has not been reported previously. Furthermore, PYP may also represent a class of proteins that bind conjugated molecules and interact with a secondary receptor system.
Photoactive yellow protein (PYP), a yellow-colored protein with a reversible photocycle, has been isolated from Ectothiorhodospira halophila (1, 2). E. halophila is an obligate anaerobe utilizing reduced sulfur compounds as electron donors for photosynthesis (3). It tolerates the high salt and high temperature conditions found in its native habitat of evaporating bodies of salt water (3). PYP has photocycle kinetics similar to those of sensory rhodopsin (4, 5); both are immediately bleached by light, followed by further bleaching on a millisecond time scale, and finally recoloring in the second time scale (2, 4). The initial bleach results from rearrangement, probably photoisomerization, of the chromophore as it is excited. The subsequent millisecond timescale event is a rearrangement of the chromophore and protein to a metastable state, which relaxes with a half-life of about 0.5-1 sec, depending upon the exact conditions (4). PYP is a water-soluble 13-kDa protein, unlike sensory rhodopsin, which is membrane-bound. The results presented here characterize PYP as a member of a recently discovered class of proteins, all with a similar /3fold, that bind small, conjugated, mostly hydrophobic molecules (6). Other proteins of this class whose structures have been determined include fatty acid-binding protein (FABP) (7), P2 myelin protein (8), serum retinol-binding protein (RBP) (9), insecticyanin (10), bilirubin-binding protein (11, 12), and /3-
METHODS PYP was purified from E. halophila strain BN9626 by using ion-exchange chromatography (1). Crystals were grown by vapor diffusion against 68% saturated ammonium sulfate (pH 7.0). The space group is P63 with unit cell dimensions a = b = 66.9, c = 40.8 A and with one molecule per asymmetric unit (2). X-ray data were collected at 20'C with an Elliott GX-21 generator source (Cu Ka, Ni filtered) and a Nicolet Imaging Proportional Counter Area Detector, using 0.20° steps in omega, a 12-cm crystal-to-detector distance, and a swing angle of 22.50. Frames were indexed and reduced by using the XENGEN package of software written by Howard et al. (18). Statistics for the native data are listed in Table 1. Heavy atom derivatives were produced by soaking crystals in solutions of the desired compound (Table 2). Data were collected as for the native crystals, but the temperature was lowered to -100C, which greatly reduced the decay problems typical for derivative crystals. Derivative difference Patterson maps were solved by inspection, and heavy atom positions were refined with the program HEAVY (19). Five derivative data sets were used (Table 2), including two lanthanides (Er, Gd) that bound at a common site, a Pt derivative, a double soak of a lanthanide and Pt, and a higher occupancy Er derivative with more sites, prepared by transferring the crystals from high salt to a 40% (wt/wt) polyethylene glycol Mr 8000 solution with 50 mM Er(CH3COO)3. All other derivatives were prepared in 80% saturated ammonium sul-
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Abbreviations: PYP, photoactive yellow protein; MIR, multiple isomorphous replacement; FABP, fatty acid-binding protein; RBP, retinol-binding protein.
6533
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Biochemistry: McRee et al.
Table 1. Statistics for native x-ray diffraction data of PYP Upper limit, A Shell of resolution 3.8 3.0 2.6 2.4 Total No. of observations 9602 8682 6522 58% 30,702 No. of unique reflections 1064 1026 998 914 4,002 No. of unique reflections possible 1068 1026 1030 1014 4,138 Average I/cr(I) 96.1 66.4 40.5 30.9 63.4 2.5 3.2 4.3 5.3 3.6 Rsym Space group P63, a = b = 66.9, c = 40.8 A. Data were collected on a Nicolet X-200A area detector. I/ur(I) is observation intensity divided by the standard deviation of I. Rsym is [i(IjFj - Favgl/Favg)/n] x 100 of all symmetry-related observations.
fate with the addition of either 0.1 mM K2PtCl4 for 7 hr or 50 mM Er(CH3COO)3 or 50 mM Gd(SO4) for 3 days. The double derivative was prepared by first soaking in an Er(CH3COO)3 solution for 3 days, after which K'PtCI4 was added for 7 more hr of soaking. The relative z translation of the Pt and lanthanide binding sites was found by difference Fourier maps using Pt single isomorphous replacement phases, including anomalous data to determine the hand. (Any derivative with a single site, or several sites with the same z coordinate, in this polar space group gives centrosymmetric phases; however, the anomalous component contains hand information that breaks the centrosymmetry.) MIR phases calculated by using the heavy atom positions shown in Table 2 gave a figure of merit of 0.70 at 2.8 A. One cycle of solvent-flattening and noise-filtering using the method of Wang (20) was applied with the envelope step modified to place points of high density in the solvent region that are contiguous with density in the protein region back into the protein region. This procedure increased the figure of merit to 0.86 with an R factor of 0.15 between the observed amplitudes and those calculated from the inverted map and a total phase shift of 260. To allow for bound solvent, a bulk solvent content of 32% was assumed, which is somewhat less than the total solvent content of 38% calculated from the molecular weight of PYP and average density of proteins. Fourier maps made with flattened and nonflattened MIR phases were fit with the computer-graphics programs GRINCH (21) and FRODO (22) to create the initial model. The model was improved by iterative cycles of refitting to electron density maps calculated with nonsolvent-flattened MIR phases combined with calculated phases to reduce model bias (23). The coefficients used were mcomb[Fo + Q(Fo - D x Fc)], where D is dependent upon the estimated error in the model as suggested by Read (24), and Q = log(mmR)/[log(mcalc) + log(mMJR)], where m is figure of merit (25). Q is 1.0 if no MIR data are present, and Table 2. Refined heavy atom parameters of PYP derivatives Derivative Site OccuX no. Y compound pancy 1. Er(CH3COO)3 1 0.3 0.509 0.109 2. Gd(SO4)3 1 0.3 0.511 0.108 3. K2PtCl4 1 0.4 0.239 0.071 4. Double soak 1 0.5 0.240 0.073
Proc. Natl. Acad. Sci. USA 86 (1989)
D is typically about 0.9. The combined figure of merit for the model and MIR phases started at 0.75 and rose to 0.79. Between cycles, the model was refined by reciprocal space methods using the program XPLOR (26). No solvent molecules were included in the model.
RESULTS AND DISCUSSION The crystallographic structure of PYP was solved by x-ray diffraction using MIR techniques. Native data were collected to 2.4 A resolution with an average I/o(I) of 63 and an overall Rsym of
