Regulation of the Thermoalkaliphilic F1-Atpase from Caldalkalibacillus Thermarum

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum Scott A. Fergusona, Gregory M. Cooka,b, Martin G. Montgomerya, Andrew G. W. Lesliec, and John E. Walkera,1 aMedical Research Council Mitochondrial Biology Unit, Cambridge Biomedical Campus, Cambridge CB2 0XY, United Kingdom; bDepartment of Microbiology and Immunology, University of Otago, Dunedin 9016, New Zealand; and cMedical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom Contributed by John E. Walker, July 25, 2016 (sent for review June 19, 2016; reviewed by Thomas M. Duncan and Dale B. Wigley) e The crystal structure has been determined of the F1-catalytic domain the F1-domains from E. coli (19, 20), but the isolated -subunit of the F-ATPase from Caldalkalibacillus thermarum, which hydro- remains in a down conformation even when ATP is not bound to lyzes adenosine triphosphate (ATP) poorly. It is very similar to those it (14–16). Deletion of its five C-terminal amino acids diminished of active mitochondrial and bacterial F1-ATPases. In the F-ATPase respiratory growth (21), but deletion of the C-terminal domain from Geobacillus stearothermophilus, conformational changes in had no growth phenotype (22). the e-subunit are influenced by intracellular ATP concentration and The F-ATPases from the thermoalkaliphile Caldalkalibacillus membrane potential. When ATP is plentiful, the e-subunit assumes a thermarum (23) from mycobacterial species (24, 25) and from “down” state, with an ATP molecule bound to its two C-terminal alkaliphilic Bacilli (26, 27) exemplify classes of eubacterial enzymes α-helices; when ATP is scarce, the α-helices are proposed to inhibit with F-ATPases that can synthesize ATP but show extreme latency ATP hydrolysis by assuming an “up” state, where the α-helices, in hydrolyzing ATP, although a weak ATPase activity can be α β devoid of ATP, enter the 3 3-catalytic region. However, in the stimulated artificially in vitro (23–27). The modes of inhibition of Escherichia coli enzyme, there is no evidence that such ATP binding the hydrolytic activity is not understood, although it has been to the e-subunit is mechanistically important for modulating the suggested that the C. thermarum enzyme is inhibited by the ’ enzyme s hydrolytic activity. In the structure of the F1-ATPase from γ-subunit having adopted a modified structure (28). The γ-subunit C. thermarum, ATP and a magnesium ion are bound to the α-helices isakeycomponentoftheenzyme’s rotor. in the down state. In a form with a mutated e-subunit unable to As described here, we have investigated possible mechanisms of bind ATP, the enzyme remains inactive and the e-subunit is down. inhibition of the F1-ATPase from C. thermarum by studying the Therefore, neither the γ-subunit nor the regulatory ATP bound to structure and properties of a version lacking the δ-subunit (re- the e-subunit is involved in the inhibitory mechanism of this partic- ferred to as F -ATPase); the δ-subunit is part of the enzyme’s α β 1 ular enzyme. The structure of the 3 3-catalytic domain is likewise stator, and has no direct role in the mechanism or regulation of closely similar to those of active F1-ATPases. However, although the ATP hydrolysis. We have reinvestigated the proposal that, in the β “ ” E-catalytic site is in the usual open conformation, it is occupied by inhibited enzyme, the γ-subunit has a modified structure, and have the unique combination of an ADP molecule with no magnesium ion studied a form of the enzyme with two mutations in a region of the and a phosphate ion. These bound hydrolytic products are likely to e-subunit where the proposed regulatory ATP molecule is bound be the basis of inhibition of ATP hydrolysis. in E. coli and G. stearothermophilus (17, 29). Our study shows that neither the γ-subunit nor the regulatory ATP bound to the Caldalkalibacillus thermarum | F1-ATPase | structure | inhibition | regulation Significance he F-ATPases (F1Fo-ATP synthases) from chloroplasts, Adenosine triphosphate (ATP), the fuel of life, is produced by a Tmitochondria, and eubacteria have evolved different ways molecular machine consisting of two motors linked by a rotor. One of regulating ATP hydrolysis (1). During darkness, chloroplast generates rotation by consuming energy derived from oxidative F-ATPases use a redox inhibitory mechanism (2, 3). Mitochon- metabolism or photosynthesis; the other uses energy transmitted drial enzymes bind an inhibitor protein called IF1, inhibitor of by the rotor to put ATP molecules together from their building – α F1-ATPase (4 7), and -proteobacterial F-ATPases have a related blocks adenosine diphosphate and phosphate. In many species the ζ – inhibitor protein called the -subunit (8 10). Many eubacterial machine is easily reversible, and various different mechanisms to F-ATPases synthesize ATP in the presence of oxygen and, under regulate the reverse action have evolved so that it is used only anaerobic conditions, hydrolyze ATP made by substrate-level when needed. In some eubacterial species, including the phosphorylation to generate the proton-motive force, which is thermoalkaliphile Caldalkalibacillus thermarum, although evidently required for maintaining cell viability in the absence of growth. constructed in a similar way to reversible machines, the reverse When the proton-motive force and cellular ATP concentration are action is severely impeded, evidently because the products of hy- low, an inhibitory mechanism that may operate to prevent ATP drolysis remain bound to the machine. wastage has been demonstrated in vitro for the F-ATPases from Escherichia coli (11), Geobacillus stearothermophilus (12), and Author contributions: J.E.W. designed research; J.E.W. supervised the project; S.A.F., G.M.C., Thermosynechococcus elongatus (13) involving their e-subunit. and M.G.M. performed research; S.A.F., G.M.C., M.G.M., A.G.W.L., and J.E.W. analyzed data; and M.G.M. and J.E.W. wrote the paper. This subunit, a component of the rotor of the enzyme, is folded into β α Reviewers: T.M.D., State University of New York Upstate Medical University; and D.B.W., an N-terminal nine-stranded -sandwich and a C-terminal -helical Imperial College London. hairpin (13–18). The β-sandwich binds the subunit to the γ-subunit The authors declare no conflict of interest. and to the c ring in the membrane domain, and the α-helices adopt Freely available online through the PNAS open access option. two conformations, “down” and “up.” In the down state of the α e Data deposition: The crystallography, atomic coordinates, and structure factors reported F-ATPase from G. stearothermophilus,the -helices of the -subunit in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes bind an ATP molecule and are associated with the β-sandwich 5HKK and 5IK2). (17); in the absence of bound ATP, the α-helices assume the up 1To whom correspondence should be addressed. Email: [email protected]. α β position, interacting with the 3 3-domain and inhibiting ATP This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hydrolysis (18). Up positions have been captured in structures of 1073/pnas.1612035113/-/DCSupplemental. 10860–10865 | PNAS | September 27, 2016 | vol. 113 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1612035113 Downloaded by guest on September 30, 2021 e-subunit is involved in the inhibitory mechanism, and that as a phosphate ion at an occupancy of 100% (Fig. S2). An ATP probably ATP hydrolysis is prevented by hydrolysis products molecule with a magnesium ion is bound in the C-terminal bound to a catalytic subunit. α-helical domain of the e-subunit. The mutant F -ATPase complex from C. thermarum was solved Results 1 by molecular replacement using the α3β3-domain from the wild- Structure Determination. The purified wild-type and mutant type structure with data to 2.6-Å resolution (Table S1). Like the F1-ATPases from C. thermarum consist of the α-, β-, γ-, and e-subunits wild-type structure, the asymmetric unit of the mutant enzyme (Fig. S1). The ATP hydrolysis activity of the wild-type and mutant contains two copies of the complex, complexes 1 and 2, with F -complexes was activated by lauryldimethylamine oxide [LDAO; electron densities of similar quality. The model of complex 1 1 α – α – – α – 0.1% (vol/vol)] to a specific activity of 33–38 U/mg protein for contains residues E,27 501; TP,26 395, 403 501; DP,26 398, – β – β – β – γ – e – both enzymes. The crystal structure of the wild-type enzyme (Fig. 1 401 502; E,1 462; TP,1 462; DP,2 462; ,2 286; and ,3 134. A and B) was solved by molecular replacement with data to 3.0-Å The resolved regions of complex 2 are almost identical, and the model contains the following residues: αE,27–501; αTP,26–394, resolution. The asymmetric unit contains two copies of the com- – α – – β – β – β – plex. The electron density for one copy was slightly better than for 403 501; DP,24 399, 403 502; E,1 462; TP,1 462; DP,1 462; γ,2–286; and e,1–134. The occupancy of nucleotides in the α-and the other, but their structures are very similar, with an rmsd in β-subunits was the same as in the wild-type structure, with full main-chain atoms of 0.37 Å. Data processing and refinement occupancy of the ADP in the β -subunit. Water molecules were statistics for both structures are summarized in Table S1.Thefinal E built into the mutant structure but, because of its relatively modest model of the better-defined copy of the wild-type complex (com- resolution, only in some instances in the wild-type structure.

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