Recombinant Streptomyces coelicolor ATP synthase subunit c (atpE)

Introduction to Streptomyces coelicolor and ATP Synthase

Streptomyces coelicolor stands as one of the most extensively studied species within the actinomycete phylum, renowned for its complex developmental cycle and remarkable capacity to produce antibiotics and other bioactive compounds. This soil-dwelling, Gram-positive bacterium has garnered significant scientific interest not only for its pharmaceutical potential but also as a model organism for studying bacterial differentiation and secondary metabolism. S. coelicolor exhibits a complex life cycle involving the formation of substrate mycelium, aerial mycelium, and ultimately spores, with each developmental stage regulated by intricate molecular and metabolic networks . The bacterium's genome has been fully sequenced, revealing approximately 8 million base pairs encoding over 7,800 genes, making it one of the largest bacterial genomes characterized to date.

ATP synthase represents a fundamental enzymatic complex present across all domains of life, serving as the primary machinery for ATP production through oxidative phosphorylation. This remarkable molecular motor harnesses the energy of electrochemical gradients to synthesize ATP, the universal energy currency of cells. In bacteria like S. coelicolor, F-type ATP synthases consist of two major components: the membrane-embedded F0 sector responsible for proton translocation across the membrane, and the catalytic F1 sector where ATP synthesis occurs. The F0 sector typically comprises several subunits, with subunit c playing a particularly crucial role in forming the proton-conducting ring structure essential for energy conversion.

The atpE gene in S. coelicolor encodes the ATP synthase subunit c protein, a small but vital component of the F0 sector. This highly hydrophobic protein spans the membrane multiple times and oligomerizes to form a ring-like structure. The significance of studying atpE extends beyond basic understanding of cellular energetics, as ATP synthase components have emerged as potential targets for antimicrobial development given their essential nature and structural differences from human counterparts.

Significance in Bacterial Physiology

The ATP synthase complex, including the c subunit, plays a pivotal role in bacterial energy metabolism, directly influencing growth, survival, and various cellular processes. In S. coelicolor, energy generation is intricately linked to its complex developmental cycle and secondary metabolite production. Understanding the structure and function of individual components like atpE may provide insights into the energetic requirements of different developmental stages and how energy metabolism interfaces with antibiotic production pathways.

Evolutionary Conservation

ATP synthase represents one of the most evolutionarily conserved protein complexes, with subunit c showing remarkable structural and functional conservation across diverse bacterial species. This conservation highlights the fundamental importance of this protein in cellular energetics and suggests that insights gained from studying S. coelicolor atpE may have broader implications for understanding bacterial energy metabolism across various species.

Protein Structure and Domains

While the three-dimensional structure of S. coelicolor atpE has not been directly determined, structural insights can be inferred from homologous proteins in other bacterial species. Typically, ATP synthase c subunits form a cylindrical ring comprising 8-15 subunits (depending on the species), with each subunit contributing to the formation of the proton channel through the membrane. Each c subunit generally contains two transmembrane α-helices connected by a short polar loop region, with the conserved acidic residue (usually glutamate or aspartate) in the second helix participating directly in proton translocation.

Analysis of the S. coelicolor atpE sequence reveals characteristic features of F-type ATP synthase c subunits, including hydrophobic stretches likely corresponding to transmembrane domains and conserved residues important for function. The protein's relatively small size and hydrophobic nature present challenges for structural studies but are consistent with its role as a membrane-embedded component of the ATP synthase complex.

Post-translational Modifications

While the search results do not specifically mention post-translational modifications of atpE in S. coelicolor, it's noteworthy that in source , a comprehensive analysis of pupylation (a prokaryotic ubiquitin-like protein modification) in S. coelicolor was conducted. The study identified 20 pupylated proteins across various functional categories, although atpE was not among them . This suggests that atpE may not undergo pupylation, at least under the conditions tested in that study.

Comparison with Related Proteins

The ATP synthase c subunit belongs to a highly conserved family of proteins present across bacteria, archaea, and eukaryotes. Sequence alignment with homologous proteins from other species would likely reveal conservation of key functional residues, particularly those involved in proton translocation. The evolutionary conservation of this protein underscores its fundamental importance in cellular energetics across diverse life forms.

Production of Recombinant atpE

The commercial availability of recombinant S. coelicolor atpE protein indicates successful development of expression and purification protocols for this challenging membrane protein. According to the available information, the recombinant protein encompasses the full-length sequence (amino acids 1-76) and includes an N-terminal His-tag to facilitate purification .

Expression Systems

The successful expression of atpE in E. coli suggests optimization of expression conditions to overcome these challenges, potentially including the use of specialized E. coli strains, controlled induction protocols, or the addition of solubilizing agents. The inclusion of an N-terminal His-tag further indicates a design strategy for subsequent affinity purification.

Purification and Quality Control

Following expression, the recombinant atpE protein can be purified to greater than 90% purity as determined by SDS-PAGE analysis . The N-terminal His-tag allows for affinity chromatography purification, typically using nickel or cobalt resins with high affinity for histidine residues. This purification approach can yield significant quantities of protein suitable for various research applications.

After purification, the protein is formulated in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 and is subsequently lyophilized for storage stability . The addition of trehalose, a disaccharide known for its protein-stabilizing properties, helps maintain protein integrity during the lyophilization process and subsequent storage.

Functional Roles of ATP Synthase Subunit c

Although the search results do not provide specific experimental data on the function of S. coelicolor atpE, its role can be inferred from the well-established functions of ATP synthase c subunits in other organisms and the high degree of conservation across species.

Role in ATP Synthesis

The c subunit forms a critical component of the F0 sector of ATP synthase, assembling into a ring-like structure embedded in the membrane. This c-ring rotates in response to proton flow through the membrane, driven by the proton motive force. The rotation of the c-ring is mechanically coupled to the γ subunit of the F1 sector, inducing conformational changes that catalyze ATP synthesis from ADP and inorganic phosphate.

Each c subunit typically contains a conserved acidic residue that participates in proton translocation. During rotation, this residue alternately accepts and releases protons, effectively converting the energy of the proton gradient into mechanical rotation. The precise stoichiometry of the c-ring (number of c subunits per ring) can vary across species and affects the bioenergetic efficiency of ATP synthesis.

Interactions with Other ATP Synthase Components

The c-ring interfaces with several other components of the ATP synthase complex. It interacts with the a subunit to form the proton channel, with the b subunit that connects to the F1 sector, and with the γ subunit that transmits rotational energy to the catalytic sites. These interactions are crucial for the assembly and function of the complete ATP synthase complex.

Potential Role in Bacterial Physiology

Beyond its direct role in ATP synthesis, the ATP synthase complex, including the c subunit, may participate in broader aspects of bacterial physiology. In some bacteria, ATP synthase has been implicated in pH homeostasis, adaptation to environmental stresses, and even virulence in pathogenic species. In S. coelicolor, with its complex developmental cycle and secondary metabolism, the energy-generating function of ATP synthase likely supports various physiological processes, potentially including antibiotic production and morphological differentiation.

Research Applications and Implications

The availability of recombinant S. coelicolor atpE protein opens various avenues for research applications, spanning from structural studies to functional analyses and potential biotechnological uses.

Functional Analyses

Recombinant atpE can be used in functional studies to investigate its properties in isolation or in reconstituted systems. Such studies might include proteoliposome reconstitution to assess proton translocation, binding assays to characterize interactions with inhibitors or other ATP synthase components, or mutagenesis studies to identify residues critical for function.

Potential Antimicrobial Applications

ATP synthase components, including the c subunit, have emerged as potential targets for antimicrobial development due to their essential nature and structural differences from mammalian counterparts. Understanding the structure and function of S. coelicolor atpE could contribute to the development of inhibitors targeting mycobacterial or other actinobacterial ATP synthases, which might have applications against pathogenic relatives like Mycobacterium tuberculosis.

Biotechnological Applications

Beyond basic research and potential therapeutic applications, ATP synthase components have been explored for various biotechnological uses. These include the development of bionanosensors, bioenergy applications harnessing the ATP-generating capacity of the complex, or the use of the c-ring as a template for designing synthetic molecular rotors.

Relationship to Other Streptomyces coelicolor Proteins

While the search results provide limited information specifically about atpE interactions with other S. coelicolor proteins, they do offer insights into other proteins and systems that may indirectly relate to ATP synthase function.

Potential Connection to Metabolic Networks

In source and , the small ORF trpM is described as stimulating growth and antibiotic production in S. coelicolor. The studies indicate that trpM over-expression causes "an over-representation of factors involved in protein synthesis and nucleotide metabolism" and a "down-representation of proteins involved in central carbon and amino acid metabolism" . While not directly linked to atpE, these findings highlight the intricate connections between energy metabolism (where ATP synthase plays a central role) and other cellular processes including antibiotic production.

Post-translational Modification Networks

Source describes pupylation, a post-translational modification in Streptomyces that tags proteins for proteasomal degradation. Although atpE was not identified among the pupylated proteins, the study revealed modification of several metabolic enzymes, chaperones, and structural proteins . This suggests sophisticated regulatory networks controlling protein turnover in S. coelicolor, which could potentially influence ATP synthase component levels under certain conditions.