Multi-subunits ATPases

ATP synthases form an evolutionarily related family of energy-coupling, ion-transporting enzymes that is responsible for the synthesis of most cellular ATP in plants, animals, and many bacteria. ATP synthases function as dual-engine rotary motors. A membrane-embedded complex (F0) acts as a turbine to transport ions (protons, but Na+ in some bacteria). A peripheral stator stalk and a central rotor stalk connect F0 to an extrinsic complex (F1) in which rotation of the asymmetric central stalk coordinates ATP synthesis or hydrolysis.

For over 30 years, the ATP synthase of the gram negative bacterium Escherichia coli (EcF1) has provided the predominant system for classical genetics and mutagenesis studies of the functional mechanism of ATP synthases. Bacterial ATPases have also been recognized as a novel and powerful target for the development of anti-bacterial drugs. A new class of effective anti-tuberculosis drugs that kills mycobacteria specifically inhibits the F0 subunit (Andries et al, 2005; Koul et al, 2007). Despite the growing importance of this multi-subunit enzyme in biology and medicine, the 3D-structure of the prototypical EcF1 enzyme has remained elusive. EcF1, a multi-protein complex formed by 9 proteins (α3, β3, γ, ε, δ), is in fact highly resistant to crystallization and has always failed to yield high quality crystals despite the effort of many laboratories worldwide.

 E.coli F1 ATPase structure determination

Figure 1. Pipeline of structure detemination used to solve the structure of the E.coli F1 ATPase catalytic core

In collaboration with Dr. Thomas Duncan, at SUNY Upstate Medical University, we have identified a stable and enzymatically active 8-protein catalytic core of EcF1 (EcF1-δ) (Fig. 1A). We crystallized EcF1-δ using a combination of robotic and manual crystallization techniques (Fig. 1B, C) and were able to collect complete diffraction data at NSLS beamline X25 and X6A. EcF1-δ crystals belong to space group C2 with unit cell parameters a=435.97Å, b=183.06Å, c=225.39Å and β-angle = 108.99°; the asymmetric unit contains four EcF1-δ complexes (~18,500 residues!). The 3D-structure of EcF1-δ was solved at 3.2Å resolution and refined to an Rwork/Free ~24.3/26.4%. This work was published in the 2011 June issue of Nature Structural and Molecular Biology (Cingolani & Duncan, 2011). The general architecture of EcF1 is analogous to that of mitochondrial F1 (Abrahams et al, 1994) and is illustrated in Fig. 1D,E. The structure contains an hexameric ‘catalytic ring’ of α- and β-subunits surrounding the upper region of a ‘central stalk’, which consists of γ and ε-subunits. Nucleotide binding sites on β subunits are responsible for ATP synthesis/hydrolysis, while sites on α subunits are noncatalytic.
Epsilon-subunit regulationFigure 2. Structure and dynamics of the epsilon subunit of the E.coli F1 ATPase catalytic core

Perhaps the most striking and novel feature of the EcF1 structure lies in the C-terminal domain of subunit ε (ε CTD), that adopts a highly extended conformation, deeply inserted into the central cavity of the enzyme (Fig. 1D). This extended conformation of the ε-subunit contrasts with ε’s homolog in MtF1 structures (Gibbons et al, 2000) and with the compact state observed for isolated E. coli ε-subunit (Uhlin et al, 1997; Wilkens & Capaldi, 1998; Yagi et al, 2007) (Fig. 2B). The extended conformation of ε-CTD in EcF1 engages both γ-subunit and catalytic ring in extensive contacts that are incompatible with functional rotation. While the extended conformation of εCTD visualized crystallographically agrees with previous chemical labeling and cross-linking studies (Wilkens & Capaldi, 1998), this structure provides a rational explanation for the inhibitory role of ε-subunit, which is well documented for ATP synthases of bacteria and chloroplasts. Regulation of ATP synthase activity by ε-subunit is however not present in the mitochondrial enzyme. As functional ATP synthase is critical for the viability of pathogenic bacteria such as Streptococcus pneumonia, Mycobacterium tuberculosis, differences in structural complexity and regulation between bacterial and mitochondrial ATP synthases can be exploited to selectively inhibit pathogenic bacteria. Thus, the structure of the autoinhibited EcF1 provides the structural basis to understand ε-subunit mediated inhibition of rotary catalysis and gives us a rational framework for developing antimicrobial agents that selectively mimic or stabilize the ε-inhibited state of bacterial ATP synthases but do not inhibit mitochondrial ATP synthases.

In the future, we will continue the structural characterization of the bacterial ATPases with the following specific aims:

  • To determine the structure of the EcF1 in the uninhibited conformation
  • To visualize the conformation of ε-subunit in the Ec-F0F1 holoenzyme.
  • To map structurally the conformation of ε-subunit in F1 ATPases of pathogenic bacteria.