Recombinant Staphylococcus aureus GTPase Era (era)

What is Staphylococcus aureus GTPase Era and what is its functional significance?

Staphylococcus aureus GTPase Era belongs to the family of deeply conserved proteins that are critically required for the assembly of bacterial-type ribosomes across species from Escherichia coli to higher organisms . Era functions as a molecular switch that cycles between different conformational states depending on its nucleotide-binding status (apo, GDP-bound, or GTP-bound) . This switching behavior is essential for proper ribosome assembly, particularly for the maturation of the small ribosomal subunit (SSU) . In bacteria like S. aureus, Era is considered essential for viability, making it not only a fundamental component of cellular machinery but also a potential target for antimicrobial development against this significant human pathogen . S. aureus presents major clinical challenges due to its ability to develop antibiotic resistance, as evidenced by methicillin-resistant S. aureus (MRSA) strains .

How is Era GTPase structurally organized?

Era GTPase consists of two principal domains with highly conserved architecture across bacterial species. The N-terminal domain is a ~170 amino acid-long G-domain with a characteristic TRAFAC GTPase fold featuring a 6-stranded β-sheet surrounded by 5 α-helices in a highly conserved alternating pattern . This domain contains five diagnostic motifs involved in GTP binding and hydrolysis, with all β-strands being parallel except for β2, which is uniquely antiparallel in all TRAFAC GTPases . The C-terminal domain is a smaller KH (K homology) domain responsible for RNA binding, particularly interaction with the 16S rRNA of the small ribosomal subunit . In some bacterial clades, this basic two-domain architecture is extended by additional domains, including YbeY, CS, SGS, DUF916, or RNase III domains, which may confer specialized functions in different organisms . The orientation between these two domains is dynamic and changes based on nucleotide binding status, directly affecting RNA binding capacity .

How does S. aureus Era compare with Era proteins from other bacterial species?

While the search results don't provide S. aureus-specific Era comparisons, the evolutionary conservation of Era across bacterial species suggests significant structural and functional similarities. Era belongs to a deeply conserved family of GTPases with structures solved from phylogenetically distant bacterial clades including E. coli, Thermus thermophilus, and Aquifex aeolicus . All bacterial Era proteins share the characteristic two-domain architecture comprising the G-domain and KH domain, though variations in exact sequence and potential regulatory mechanisms may exist between species . Considering S. aureus's clinical importance and unique evolutionary adaptations, researchers should note that while core Era functions are likely conserved, species-specific interactions with other cellular components might exist. Given S. aureus's remarkable ability to adapt and evolve, as evidenced by approximately 22% of its genome being non-coding and variable between different strains, Era-related pathways might similarly show strain-specific adaptations .

What is the mechanism of Era's conformational switching?

Era's conformational switching represents a fundamental aspect of its cellular function and involves coordinated structural changes across the protein. Like other TRAFAC GTPases, Era operates as a molecular switch rather than a GDP/GTP sensor, as its binding affinities (Ks values) for these nucleotides are in the low micromolar range, much less than intracellular nucleotide concentrations . X-ray crystallographic studies of bacterial Era complexes have revealed striking differences between different nucleotide-bound states . In the apo- and GDP-bound Era proteins (OFF-state), the KH domain is rotated such that a negatively charged helix αD partially blocks access to the RNA-binding groove . By contrast, in the GTP-bound state (ON-state), the KH domain is reoriented to allow unobstructed RNA access to the binding site . This nucleotide-dependent conformational change provides a mechanism for coupling GTP hydrolysis to the mechanical movement required for ribosome assembly progression .

How does Era interact with ribosomal components?

Era's interaction with ribosomal components, particularly the small subunit (SSU) rRNA, is central to its function in ribosome assembly. The KH domain of Era specifically recognizes and binds to the 3'-minor domain of the SSU rRNA, with this interaction being regulated by the nucleotide-binding state of the G-domain . Interestingly, there appears to be an apparent paradox in the relationship between nucleotide binding and RNA interaction. Some in vitro observations indicate that only apo-Era significantly binds to 16S rRNA and the mature SSU, whereas addition of GDP or GTP abolishes these interactions . This contrasts with the structural data suggesting that GTP binding facilitates RNA access to the binding site . Recent studies of mitochondrial Era homologue ERAL1 have found it in a nucleotide-free form on early SSU assembly intermediates, even though its conformation resembles the activated ON-state . These findings suggest a more complex, multi-state control of Era-ribosome association by nucleotides than initially thought, possibly involving recruitment of Era in the apo-state followed by later GTP acquisition during assembly .

What experimental systems are used to study Era function?

Several experimental systems have been developed to study Era function in different bacterial species, which can be adapted for S. aureus research. One key approach is the creation of Era-depleted strains to study the protein's essential functions . This typically involves genetic manipulation to place the era gene under control of an inducible promoter, allowing researchers to modulate Era expression levels . For biochemical and structural studies, recombinant Era protein expression systems have been established, often using pET-based expression vectors in E. coli . Cryo-electron microscopy (cryo-EM) has proven valuable for examining Era-30S complexes, providing insights into the structural basis of Era-ribosome interactions . Additionally, in vitro reconstitution systems combining purified Era with mature 30S ribosomal subunits offer controlled environments for studying binding kinetics and conformational changes . For studying Era's GTPase activity, researchers typically employ spectrophotometric assays that measure either phosphate release or nucleotide consumption/production during GTP hydrolysis.

How does Era function within the broader context of S. aureus ribosome assembly?

Era functions as part of a complex network of assembly factors orchestrating ribosome biogenesis in bacteria, though the specific details for S. aureus remain to be fully characterized. GTPases like Era have been leveraged by bacteria to guide the biogenesis of both ribosomal subunits at specific assembly steps . Era's ability to rhythm the assembly process through its GTPase cycle provides a checkpoint mechanism ensuring proper maturation of the small ribosomal subunit . Within S. aureus, Era likely cooperates with other assembly factors and ribosomal proteins in a hierarchical assembly pathway. S. aureus's remarkable adaptability and genetic variation (approximately 22% of its genome is non-coding and can differ between strains) suggests that ribosome assembly pathways, including Era's exact role, might show strain-specific adaptations . Understanding these species-specific aspects of Era function in S. aureus ribosome assembly would require dedicated studies comparing S. aureus Era with homologs from model organisms like E. coli where ribosome assembly is better characterized. Such studies would be particularly relevant given S. aureus's clinical importance and the potential of ribosome assembly pathways as antimicrobial targets.

How can researchers express and purify recombinant S. aureus Era?

Expression and purification of recombinant S. aureus Era requires careful optimization of multiple parameters to obtain functional protein suitable for downstream applications. Based on existing protocols for Era proteins, researchers can use E. coli-based expression systems with vectors like pET15b, which has been successfully employed for Era expression . The gene encoding S. aureus Era can be PCR-amplified from genomic DNA using primers designed based on the annotated S. aureus genome sequence. After cloning into an appropriate expression vector with a fusion tag (typically His6 for ease of purification), expression conditions need optimization, with special attention to temperature, inducer concentration, and duration to maximize soluble protein yield. For purification, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is typically the first step for His-tagged proteins, followed by size exclusion chromatography to remove aggregates and obtain homogeneous protein preparations. Assessment of protein quality through SDS-PAGE, Western blotting, and activity assays (GTPase activity) is essential before proceeding to functional studies. For structural studies or assays requiring nucleotide-free Era, additional steps to remove bound nucleotides may be necessary, such as treatment with alkaline phosphatase or extensive dialysis against chelating agents.

What approaches can be used to study Era-depletion effects in S. aureus?

Studying Era-depletion effects in S. aureus requires genetic tools to create conditional mutants of this essential gene. One effective approach is to generate an Era-depleted strain by placing the native era gene under control of an inducible promoter, similar to methods described for E. coli . This can be achieved by inserting the S. aureus era gene at a different chromosomal locus under control of an inducible promoter (like the arabinose-inducible araBAD promoter), followed by deletion or disruption of the native era gene . Temperature-sensitive plasmids like pSim6 can facilitate homologous recombination for chromosomal integration . Once established, these conditional strains allow researchers to modulate Era expression by controlling inducer concentration, enabling studies of cellular effects during Era depletion. Phenotypic characterization should include growth curves, microscopic examination of cell morphology, and ribosome profile analysis using sucrose gradient centrifugation to quantify free ribosomal subunits versus assembled ribosomes. RNA-seq and proteomics analyses can provide insights into global gene expression changes during Era depletion. Additionally, genetic suppressor screens may identify genes or pathways that can partially compensate for Era deficiency, potentially revealing functional networks involving Era in S. aureus.

What techniques are suitable for studying Era-ribosome interactions?

Multiple complementary techniques can provide insights into Era-ribosome interactions in S. aureus. Cryo-electron microscopy (cryo-EM) has emerged as a powerful tool for visualizing Era-ribosome complexes, as demonstrated by recent studies of Era homologs . Sample preparation typically involves incubating purified recombinant Era with isolated ribosomal subunits or pre-ribosomal particles in appropriate buffer conditions . Filter binding assays and gel shift assays offer quantitative approaches to measure binding affinities between Era and ribosomal components under different nucleotide conditions. Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) can provide real-time binding kinetics data. For identifying specific RNA sequences recognized by Era, techniques like RNA footprinting, CLIP-seq (cross-linking immunoprecipitation followed by sequencing), or hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map interaction sites at nucleotide resolution. Fluorescence-based techniques, including Förster resonance energy transfer (FRET) using fluorescently labeled Era and ribosomal components, can monitor conformational changes during binding events. In vivo approaches like ribosome profiling in Era-depleted versus wild-type S. aureus can provide insights into Era's impact on global translation and ribosome biogenesis under physiological conditions.

How can researchers overcome protein solubility and stability issues with recombinant S. aureus Era?

Researchers working with recombinant S. aureus Era may encounter solubility and stability challenges that require systematic troubleshooting approaches. Expression temperature optimization is crucial, with lower temperatures (16-20°C) often favoring proper folding over rapid expression. Testing different E. coli expression strains, particularly those designed for membrane or toxic proteins (like C41/C43 or BL21-AI), can improve yield of soluble protein. Fusion tags beyond the standard His-tag, such as MBP (maltose-binding protein) or SUMO, can significantly enhance solubility, though the impact on Era activity must be verified. Buffer optimization during purification is essential, with parameters including pH (typically 7.0-8.0 for Era proteins), salt concentration (150-300 mM NaCl), and the addition of stabilizing agents like glycerol (5-10%). GTPases often benefit from the presence of magnesium ions (5-10 mM MgCl2) and sometimes low concentrations of GDP or GTP analogs in purification buffers. If inclusion bodies form despite optimization, protocols for denaturation and refolding can be attempted, though these often result in lower recovery of active protein. For long-term storage, flash-freezing aliquots in liquid nitrogen after addition of 10% glycerol and storage at -80°C typically preserves activity better than refrigeration. Before proceeding to functional assays, verification of proper folding using circular dichroism spectroscopy and GTPase activity assays is strongly recommended.

What strategies can address nucleotide binding and hydrolysis challenges in Era functional studies?

Nucleotide binding and hydrolysis studies with Era present several technical challenges that require specific strategies to overcome. When measuring GTPase activity, background hydrolysis can be minimized by using high-purity GTP preparations and including appropriate controls. For accurate activity measurements, coupled-enzyme assays that link phosphate release to NADH oxidation (which can be monitored spectrophotometrically) offer higher sensitivity than direct phosphate detection methods. Control experiments exploring the influence of potential cofactors, including potassium ions, ribosomal components, or other bacterial proteins, may reveal conditions that enhance Era's intrinsically low GTPase activity. For studying nucleotide-free Era, extensive dialysis against EDTA-containing buffers followed by size exclusion chromatography can remove bound nucleotides, though complete removal should be verified by HPLC analysis or other suitable methods. Non-hydrolyzable GTP analogs like GDPNP or GTPγS are valuable for capturing the GTP-bound state in structural studies, but researchers should be aware that these analogs may not perfectly mimic GTP in all aspects of Era function . When comparing Era from different bacterial species or mutant variants, standardized assay conditions and appropriate normalization are essential for meaningful comparisons of kinetic parameters like Km and kcat. Researchers should also be mindful of the paradoxical relationship between nucleotide binding and RNA interaction observed with Era, where apo-Era may bind RNA more strongly than nucleotide-bound forms in some contexts .