The 2.8-Å Structure of Rat Liver F1-Atpase: Configuration of a Critical Intermediate in ATP Synthesis/Hydrolysis

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11065–11070, September 1998 Biochemistry The 2.8-Å structure of rat liver F1-ATPase: Configuration of a critical intermediate in ATP synthesisyhydrolysis (ATP synthaseyFoF1-ATPaseyoxidative phosphorylationymitochondriayx-ray diffraction) MARIO A. BIANCHET*, JOANNE HULLIHEN†,PETER L. PEDERSEN†, AND L. MARIO AMZEL*‡ Departments of *Biophysics and Biophysical Chemistry and †Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185 Communicated by William A. Catterall, University of Washington School of Medicine, Seattle, WA, July 9, 1998 (received for review April 3, 1998) ABSTRACT During mitochondrial ATP synthesis, F1- different conformation from the other two, which are distinct but ATPase—the portion of the ATP synthase that contains the similar to each other (Fig. 1). This marked asymmetry was catalytic and regulatory nucleotide binding sites—undergoes proposed to be an intrinsic property of the enzyme (14). The a series of concerted conformational changes that couple structure of rat liver F1-ATPase crystals reported here (‘‘three- proton translocation to the synthesis of the high levels of ATP nucleotide structure’’), grown in the presence of substantially required for cellular function. In the structure of the rat liver higher, more physiological concentrations of nucleotides, con- F1-ATPase, determined to 2.8-Å resolution in the presence of tains nucleotides in all three b subunits and has all b subunits in physiological concentrations of nucleotides, all three b sub- highly similar conformations (Fig. 1). This structure, with all units contain bound nucleotide and adopt similar conforma- catalytic and noncatalytic sites occupied by nucleotide, is a form tions. This structure provides the missing configuration of F1 of the enzyme that provides the missing configuration of F1 necessary to define all intermediates in the reaction pathway. necessary to define all intermediates in the reaction pathway of Incorporation of this structure suggests a mechanism of ATP ATP synthesis. Inclusion of this additional, key configuration y synthesis hydrolysis in which configurations of the enzyme results in significant modifications to previously proposed mech- with three bound nucleotides play an essential role. anisms. In mitochondria and bacteria, the ATP synthase complex (F0F1) MATERIALS AND METHODS uses the electrochemical proton gradient generated by the respi- Preparation and Characterization of the Crystals. The protein ratory chain to produce ATP at the concentrations needed for was purified and crystallized by ammonium sulfate precipitation cellular functions. Homologous proteins produce ATP in chlo- in 200 mM KPi plus 5 mM ATP (pH 7.5) as described (15, 17). m roplasts by using the proton gradient generated during photo- Crystals of F1 were dissolved in 50 l of 250 mM KPi plus 5 mM synthesis. The mechanism by which ATP synthase couples the EDTA (pH 7.5), and 15 mg were subjected to SDSyPAGE in proton gradient to the synthesis of ATP remains one of the cylindrical gels. After staining with Coomassie blue, gels were central questions in bioenergetics. The F1 sector, the portion of scanned by using a scanning attachment on a Gilford spectro- the enzyme that contains the nucleotide binding and catalytic photometer. To determine the capacity of redissolved crystals to sites, can be isolated as a soluble multiple subunit protein rebind to F -depleted inner membrane vesicles of mitochondria a b gd« ' 1 ( 3 3 , molecular weight is 371,000; refs. 1 and 2) that and to regain sensitivity to oligomycin, an inhibitor of oxidative exhibits ATPase activity (F1-ATPase). The lengths of the sub- phosphorylation, purified F (200 mg) in 250 mM KP plus 5 mM a b 1 i units in the rat liver enzyme are as follows: , 510 residues; , 479 EDTA (pH 7.5) was reconstituted with F -depleted inner mem- g d « 1 residues; , 270 residues; , 142 residues; and , 50 residues. In brane vesicles (0.5 mg) of rat liver mitochondria in a 0.1-ml system negatively stained micrographs, the rat liver enzyme appears as at 25°C. Inner membrane vesicles and F1-depleted inner mem- six regions in an hexagonal arrangement surrounding a central brane vesicles were prepared as described (18). Inhibition was mass (3). During ATP synthesis, the F1 sector undergoes con- measured in the presence of 1 mg of oligomycin. To determine the certed conformational changes (the ‘‘binding change mecha- relative capacity of ATP and MgCl2 to initiate the F1 ATPase nism’’; for reviews see references 4–8) that couple changes in the reaction, kinetic measurements were made initiating the reaction nucleotide binding sites of the b subunits with rotations of the g either with 4 mM ATP or with 4.8 mM MgCl2. ATPase activity subunit (9–14). Understanding the nature of these structural was assayed by coupling the formation of ADP to the pyruvate changes is essential for understanding ATP synthesis. In this kinase and lactic dehydrogenase reactions as described (18). The paper, we report the structure of a native form of rat liver decrease in optical density of NADH at 340 nm was monitored. F1-ATPase crystallized by precipitation with ammonium sulfate Structure Determination. All data were collected at 100 K on from a buffer used for the determination of ATPase activity: 5 21 an R-Axis II Image Plate System (Molecular Structure, The mM ATP and 200 mM potassium phosphate (pH 7.2) (15). Mg Woodlands, TX) operating on a Rigaku RU200 generator im- was omitted from the buffer to prevent immediate hydrolysis of plemented with a graphite monochromator. Crystals were stabi- the ATP in the crystallization media. lized for freezing by the addition of glycerol (33–35%, wtyvol) to The structure of bovine heart F -ATPase crystallized from a 20 1 the mother liquor. Typically, 60° of data were collected from a mM MgSO solution containing the inhibitors 59-adenylyl-b,g- 4 single crystal that yielded complete data sets with high redun- imidodiphosphate (250 mM), ADP (5 mM), and azide (0.02% dancy. To check the symmetry, one set of 200° of data was wtyvol) has been reported (14, 16). This structure (hereafter the collected and processed in space group P1, in three different ‘‘two-nucleotide structure’’) contains nucleotides in only two of orientations of C2, and in R32. Data were processed by using the the three b subunits; the empty subunit has a substantially program DENZO (19) and were scaled by using SCALEPACK (19). The publication costs of this article were defrayed in part by page charge Data deposition: The atomic coordinates have been deposited in the payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Data Bank, Biology Department, Brookhaven National Lab- accordance with 18 U.S.C. §1734 solely to indicate this fact. oratory, Upton, NY 11973 (PDB ID Code 1MAB). © 1998 by The National Academy of Sciences 0027-8424y98y9511065-6$2.00y0 ‡To whom reprint requests should be addressed. e-mail: mario@ PNAS is available online at www.pnas.org. neruda.med.jhmi.edu. 11065 Downloaded by guest on September 27, 2021 11066 Biochemistry: Bianchet et al. Proc. Natl. Acad. Sci. USA 95 (1998) FIG. 1. Schematic representation of the crystallization conditions and the bound nucleotides in the two-nucleotide structure (Left, bovine heart, ref. 14) and the ‘‘three-nucleotide structure’’ (Right, rat liver, this work). The possibility of twinning was checked by calculating the ratio ^F&2y^F2& (20). All heavy atom calculations were carried out with the CCP4 suite of programs (21). Heavy atom positions were estimated from difference Patterson maps. Different heavy atom derivatives were placed on the same origin either by the presence of common sites or by the use of difference Fourier maps. Refinement of heavy atom positions and phase calculations were performed with the program MLPHARE as implemented in CCP4. Phase improvement through density modification was carried out with the program DM as implemented in CCP4. Interpretation of FIG.2. (A) Diffraction quality crystals of rat liver F1.(B) Scan of an the maps and building of the atomic models were carried out with y a b SDS PAGE gel of redissolved F1 crystals. Crystals of F1 were dissolved the program O (22). One subunit, one subunit, and part of one in 50 ml of 250 mM KPi plus 5 mM EDTA (pH 7.5), and 15 mg were g subunit were built. The g subunit, present in one-third copy per subjected to SDSyPAGE in cylindrical gels. (C) Capacity of redissolved asymmetric unit, was built by using maps contoured at densities crystals to rebind to F1-depleted inner membrane vesicles of mitochondria about one-half the density level used to contour the rest of the and regain sensitivity to oligomycin, an inhibitor of oxidative phosphor- m map. Models were refined with the program XPLOR (23). Cycles ylation. Where indicated, 1 g oligomycin was present. (D) Relative of gradient refinement were followed by runs of simulated capacity of ATP and MgCl2 to initiate the F1 ATPase reaction. The annealing using slow cooling. After convergence, the model was decrease in optical density of NADH at 340 nm was monitored. Reaction 1 was initiated with 4 mM ATP, and reaction 2 was initiated with 4.8 mM checked against the original map, 2Fo-Fc maps, and maps using MgCl2. phases and weights obtained by a combination of experimental abg d « and calculated phases by using the program SIGMAA as imple- one-third of one F1 molecule ( 1y3 1y3 1y3). Because this is an mented in CCP4. When necessary, sections of the model were unusual result, the symmetry and space group assignments were rebuilt by using the program O. Special attention was given to checked thoroughly during different stages of the structure trying to identify regions that could be built in more than one determination by using many different schemes.

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