Recombinant Acinetobacter baumannii ATP synthase subunit delta (atpH)

What is the composition of the A. baumannii F1F0-ATP synthase complex and where does subunit delta fit within this structure?

The A. baumannii F1F0-ATP synthase is composed of multiple subunits with the composition α3:β3:γ:δ:ε:a:b2:c10, forming a molecular machine essential for this strictly respiratory opportunistic human pathogen . The F1 portion, which has been successfully expressed as a recombinant complex, contains subunits α3:β3:γ:ε . The delta subunit (atpH) helps connect the F1 catalytic portion to the membrane-bound F0 portion. While studies have characterized the F1-ATPase, the delta subunit remains less extensively studied compared to other components like epsilon, which has been shown to play a critical role in regulating ATP hydrolysis.

How does the A. baumannii ATP synthase differ from ATP synthases in other bacterial species?

A. baumannii ATP synthase exhibits notably latent ATPase activity, making it incapable of ATP-driven proton translocation . This feature distinguishes it from many other bacterial ATP synthases and appears to be largely regulated by the epsilon subunit. Studies have shown that removing the epsilon subunit results in a 21.5-fold increase in ATP hydrolysis activity . Unlike some other bacterial ATP synthases where MgATP binding regulates the conformation of regulatory subunits, the A. baumannii epsilon subunit does not bind MgATP . These differences may reflect the adaptation of A. baumannii to its ecological niche and its strictly respiratory metabolism.

What expression systems have been successful for producing functional recombinant A. baumannii ATP synthase subunits?

Based on published research, E. coli expression systems have been successfully used to produce recombinant A. baumannii ATP synthase components . For the F1-ATPase complex (α3:β3:γ:ε), researchers have managed to generate and purify the first recombinant complex showing latent ATP hydrolysis activity . For expression of individual subunits, similar heterologous expression systems would likely be applicable for atpH. When expressing recombinant A. baumannii proteins in E. coli, codon optimization and careful selection of expression vectors and host strains are critical considerations, as demonstrated in studies of other A. baumannii recombinant proteins .

What purification strategies yield optimal results for A. baumannii ATP synthase subunits?

Successful purification of A. baumannii recombinant proteins has been achieved using nickel-affinity chromatography for His-tagged constructs . For ATP synthase subunits specifically, researchers purified the recombinant F1-ATPase complex with retained functionality . When purifying recombinant atpH, similar approaches combined with appropriate buffer conditions would be advisable, followed by confirmation of proper folding and activity through structural and functional assays.

How can researchers verify the structural integrity of purified recombinant atpH?

Verification of structural integrity for recombinant A. baumannii ATP synthase subunits has employed multiple complementary techniques. For the epsilon subunit, researchers have successfully used NMR to determine solution structures , revealing important domain-domain interactions. Cryo-electron microscopy at resolutions of approximately 3.0 Å has been used to visualize the architecture of the F1-ATPase complex . For recombinant atpH, circular dichroism spectroscopy could initially assess secondary structure content, followed by limited proteolysis to evaluate domain folding. Ultimate verification would involve functional reconstitution with other ATP synthase components to assess if the purified protein can participate in proper complex assembly.

What methods are appropriate for assessing the functional activity of recombinant atpH?

Since the delta subunit primarily serves a structural role in connecting F1 and F0 sectors, functional analysis would focus on:

• Binding assays with interacting partners (particularly the F1 sector components)

• Reconstitution experiments to assess if recombinant atpH can restore function in delta-depleted complexes

• Structural studies (such as cross-linking) to map interaction interfaces with other subunits

How does the ATP synthesis/hydrolysis regulatory mechanism in A. baumannii differ from other bacteria, and what implications might this have for atpH function?

A. baumannii F1F0-ATP synthase exhibits an unusual regulatory mechanism where ATP hydrolysis is naturally inhibited (latent), preventing wasteful ATP consumption . Unlike other bacterial ATP synthases where MgATP binding regulates subunit conformations, A. baumannii epsilon does not bind MgATP yet still controls ATP hydrolysis through conformational changes . This distinctive regulatory mechanism involves the C-terminal domain of epsilon undergoing structural transformation between "up" (extended) and "down" (compact) conformations .

Delta subunit presumably must accommodate these conformational changes while maintaining structural integrity of the complex. Understanding how atpH might influence or respond to these unique regulatory features could reveal pathogen-specific mechanisms. Mutational analysis targeting interaction interfaces between delta and epsilon could help elucidate how these subunits cooperate in the unique regulatory mechanism of A. baumannii ATP synthase.

What experimental approaches can distinguish between the roles of different subunits in ATP synthase assembly versus catalytic regulation?

Researchers studying the A. baumannii epsilon subunit successfully employed C-terminal truncations and point mutations to identify regions critical for ATP hydrolysis inhibition . Similar approaches could be applied to atpH, generating systematic variants to determine which regions are essential for assembly of the complex versus regulatory functions. Additionally, heterologous expression systems like those used for investigating epsilon's role in ATP synthesis could be employed to study atpH function in a cellular context .

What structural techniques have been most informative for studying A. baumannii ATP synthase components?

Research on A. baumannii ATP synthase has benefited from complementary structural techniques:

• Cryo-electron microscopy (cryo-EM) at 3.0 Å resolution has visualized the architecture of the F1-ATPase complex, revealing the extended position of the epsilon C-terminal domain .

• NMR solution structures have characterized the compact form of the epsilon subunit, providing insights into domain-domain interactions .

• Mutational studies coupled with activity assays have mapped functional regions of the regulatory subunits .

For studying recombinant atpH, a similar multi-technique approach would be valuable. Cryo-EM could position delta within the larger complex context, while NMR or X-ray crystallography of the isolated subunit could provide atomic-level details of its structure. The structural insights would inform functional studies through targeted mutations of key residues identified in the structures.

How do conformational changes in ATP synthase subunits contribute to function, and what might this suggest about atpH dynamics?

Recent cryo-EM studies of the A. baumannii F1-ATPase have revealed four distinct conformational states representing transition states during catalysis . These studies show that the C-terminal domain of epsilon undergoes substantial structural transformation, forming the switch between ATP hydrolysis "off" and ATP synthesis "on" states . These conformational changes occur in concert with altered motions and interactions in both the catalytic and rotary subunits.

What are the challenges in obtaining high-resolution structures of membrane-associated ATP synthase components like atpH?

Membrane-associated ATP synthase components present several structural biology challenges:

• Expression and purification while maintaining native conformations

• Amphipathic nature requiring specialized detergents or membrane mimetics

• Conformational heterogeneity due to dynamic functions

• Potential instability when isolated from partner subunits

While the F1 portion of A. baumannii ATP synthase has been successfully studied by cryo-EM at 3.0 Å resolution , the membrane-associated components present additional difficulties. For atpH, which interacts with both soluble and membrane components, determining conditions that stabilize its native conformation is critical. Recent advances in cryo-EM have enabled visualization of conformational states in the context of the F1 complex , suggesting this technique may be valuable for studying atpH in its native context.

How might structural insights into atpH and other A. baumannii ATP synthase components inform antimicrobial development?

The structural studies of A. baumannii F1-ATPase reveal potential pathogen-specific targets for inhibitor development . Particularly, the unique regulatory mechanisms and structural features distinguish it from human ATP synthases and those of commensal bacteria. The sites where epsilon's C-terminal domain interacts with catalytic and rotary subunits represent potential targets for inhibitors aimed at disrupting ATP synthesis or depleting ATP through dysregulated hydrolysis .

For atpH specifically, its interface with both F1 and F0 sectors may offer unique targeting opportunities. Compounds disrupting these interactions could potentially destabilize the entire complex. Since A. baumannii is strictly aerobic and depends entirely on oxidative phosphorylation , its ATP synthase represents a vulnerability that could be exploited for antimicrobial development, particularly against multi-drug resistant strains.

What in silico approaches can predict functional residues of atpH for targeted mutagenesis studies?

Researchers studying the epsilon subunit successfully identified critical residues for domain-domain formation through mutational analysis . For atpH, similar approaches combining computational prediction with experimental validation would be valuable. Homology modeling based on structurally characterized delta subunits from related organisms could predict the three-dimensional structure, while evolutionary analysis could identify conserved residues likely important for function. These predictions would inform targeted mutagenesis experiments to validate functional roles.

How does subunit stoichiometry and assembly order affect the functional properties of recombinant A. baumannii ATP synthase complexes?

Studies of the A. baumannii F1-ATPase have demonstrated that subunit composition significantly impacts enzymatic activity. Removal of the epsilon subunit resulted in a 21.5-fold increase in ATP hydrolysis activity , highlighting the regulatory importance of proper subunit assembly. For understanding atpH function, systematic reconstitution experiments with defined subunit compositions would reveal its contribution to complex stability and activity.

In vitro assembly studies could determine whether atpH associates first with the F1 or F0 sector, providing insights into the biogenesis pathway of the ATP synthase complex. Such knowledge would inform strategies for generating functional recombinant complexes for structural and biochemical studies, particularly for screening potential inhibitors targeting the assembled complex.