Regulation of the thermoalkaliphilic F ₁ -ATPase from Caldalkalibacillus thermarum

Abstract

The crystal structure has been determined of the F 1 -catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F 1 -ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the e-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the e-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α 3 β 3 -catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the e-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F 1 -ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated e-subunit unable to bind ATP, the enzyme remains inactive and the e-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the e-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α 3 β 3 -catalytic domain is likewise closely similar to those of active F 1 -ATPases. However, although the β E -catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

The NMR solution structure of Mycobacterium tuberculosis F‐ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine

“…The overall topology of Mte reveals a well conserved b-barrel for the NTD and a helix-loop-helix fold for the CTD. Both domains are connected by a short linker segment formed by the amino acids S88-E89, which was similar to the overall domain arrangement determined for subunit e of other bacterial F-ATP synthases [11][12][13][14]. The overall RMSD values between Mte and its E. coli counterpart, Ece, are about 2.3 A for 96 backbone Ca.…”

“…However, ATP does not bind to ε at a lower concentration (0.1 m m ), and moves into the ε e ‐ and inhibitory state. Such up‐movements could not be observed in the recent C. thermarum F 1 ‐ATPase structure in which a mutated ε subunit was generated, but was unable to bind ATP . In addition, Mt ε does not bind ATP and Ec ε binds ATP with a K D of about 20 m m .…”

“…As shown for the longer EceCTD, this domain can either sit on top of the eNTD (Fig. 1A), called the compact state (e c ) [11][12][13][14], or the eCTD can be extended (e e ) [7,[15][16][17], causing enzymatic inhibition. So far the solution shape of Mte (121 amino acids), derived from small angle X-ray scattering data, and the NMR structure of its very C-terminal region (amino acids) 103 DPRIAARGRARLRAVGAI 120 have been described [6].…”

The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase

“…In addition, crystal structures of the isolated ε subunit from thermophilic Bacillus PS3 [24] could give first insights into ATP binding to subunit ε. A theoretical study on the ε subunit from thermophilic Bacillus PS3 [30] could identify that Mg 2+ is bound to ATP:Oα/Oβ, being confirmed recently by the structure of the ε subunit from Caldalkalibacillus thermarum [31]; the corresponding ATP binding site of the ε subunit from Caldalkalibacillus thermarum is shown in S1 Fig.…”

Catalytic robustness and torque generation of the F1-ATPase

“…2) (Cingolani and Duncan 2011; Duncan et al 1986; Ferguson et al 2016; Menz et al 2001). Several crystal structures of F 1 -ATPases prepared in different conditions and with different ligands have been reported so far (Abrahams et al 1996; Braig et al 2000; Cabezón et al 2003; Kagawa et al 2004; van Raaij et al 1996).…”