Recombinant Acinetobacter sp. 50S ribosomal protein L31 type B (rpmE2)

Structural and Functional Characteristics

What is the primary structure and key characteristics of Acinetobacter baumannii 50S ribosomal protein L31 type B (rpmE2)?

Acinetobacter baumannii 50S ribosomal protein L31 type B (rpmE2) is a full-length protein consisting of 84 amino acids with the sequence "MRKDIHPAYQQVLFHDTNADVYFLIGSTIQQTKQTKEYPGQVYPYVTLDISSASHPFYTGEVRQASNEGRVASFNKRFARFNRKS" . The protein has a UniProt accession number of B2I2S7 and is also referenced as rpmE2 in scientific literature . Structurally, it forms part of the central protuberance of the 50S ribosomal subunit and contributes to bridge B1b, which connects the 50S and 30S subunits . The protein plays a critical role in ribosomal stability and function, particularly in the context of translation dynamics. Research has shown that the protein can become more rigid upon antibiotic binding to the ribosome, suggesting its potential involvement in antibiotic response mechanisms .

How does the recombinant form of 50S ribosomal protein L31 type B differ from its native form in Acinetobacter baumannii?

The recombinant form of the 50S ribosomal protein L31 type B is typically expressed in Escherichia coli expression systems rather than extracted from native Acinetobacter baumannii . This heterologous expression may introduce minor structural variations due to differences in post-translational modifications and folding environments between the host and native organisms. The recombinant protein maintains the full 84-amino acid sequence of the native protein, ensuring preservation of primary structure integrity . When purified, the recombinant protein shows a purity of >85% as determined by SDS-PAGE analysis, which is sufficient for most research applications . One notable difference is that the recombinant protein may include affinity tags to facilitate purification, although the specific tag type is typically determined during the manufacturing process and may vary between preparations . These differences should be considered when interpreting experimental results, especially in structural or interaction studies where subtle conformational changes might affect outcomes.

What are the optimal storage conditions for recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B to maintain its stability and activity?

For optimal storage of recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B, temperature and formulation are critical factors affecting stability. The protein should be stored at either -20°C or -80°C, with the latter preferred for long-term storage . The shelf life of the protein varies depending on its formulation: liquid preparations typically maintain stability for approximately 6 months at these temperatures, while lyophilized preparations remain stable for up to 12 months . When working with the protein, it is strongly recommended to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and activity loss; instead, researchers should prepare small working aliquots stored at 4°C for up to one week . For reconstitution of lyophilized protein, deionized sterile water should be used to achieve a concentration of 0.1-1.0 mg/mL, and addition of glycerol to a final concentration of 5-50% (with 50% being standard) is advised for long-term storage stability . These storage conditions ensure that the protein maintains its structural integrity and functional properties for the duration of experimental work.

Research Applications and Methodologies

What experimental techniques are most effective for studying the interactions between Acinetobacter baumannii 50S ribosomal protein L31 type B and antibiotics?

Cryo-electron microscopy (cryo-EM) has proven to be particularly effective for studying interactions between Acinetobacter baumannii ribosomal components and antibiotics, as demonstrated by high-resolution structures of the 70S ribosome in complex with drugs such as tigecycline and amikacin . For specific studies of L31 type B interactions, multibody refinement techniques can enhance resolution by addressing conformational heterogeneity, resulting in maps with resolution ranging from 2.5Å to 3.0Å . Computational tools like Arpeggio have been successfully applied to calculate and characterize interactions between ribosomal proteins and ligands, revealing specific carbon-pi and donor-pi interactions that may be critical for antibiotic binding . Additionally, principal component analysis has been valuable for identifying the largest variations in structural data, distinguishing between effects like 30S head rotation and intersubunit rotation that may influence L31 dynamics . When designing such experiments, researchers should consider that protein bL31 appears more well-resolved in tigecycline-bound structures compared to amikacin-bound structures, suggesting that different antibiotics may have variable effects on L31 stability and conformational rigidity .

How should researchers design controls when studying the function of recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B in ribosomal assembly?

When studying the function of recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B in ribosomal assembly, multiple control strategies must be implemented to ensure experimental validity. Researchers should include both positive controls using native ribosomal extracts containing naturally assembled L31 protein and negative controls using ribosomal preparations where L31 has been selectively depleted or mutated . Structural integrity validation is essential, comparing the conformation of the recombinant protein to established parameters; based on available data, proper L31 folding can be verified using backbone conformation metrics similar to those used for other ribosomal proteins, where Ramachandran favored percentages typically range from 90-95% . Since L31 contributes to bridge B1b that connects ribosomal subunits, controls should include intersubunit association assays comparing wild-type and L31-modified ribosomes to assess bridge formation capacity . Additionally, given that L31 appears more rigid in tigecycline-bound ribosomes, researchers might use this antibiotic as a positive control for L31 stabilization, while comparing protein dynamics in the presence and absence of antibiotics that don't directly interact with this region as an important experimental control .

What approaches should be used for reconstituting functional 70S ribosomes using recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B?

Reconstituting functional 70S ribosomes with recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B requires a carefully controlled stepwise approach. The recombinant protein should first be properly reconstituted from its lyophilized form using deionized sterile water to a concentration of 0.1-1.0 mg/mL, with brief centrifugation recommended prior to opening the vial to ensure all contents are at the bottom . For ribosome assembly experiments, researchers should begin with purified 50S subunits depleted of native L31 protein, which can be achieved through selective washing protocols or genetic knockouts in model systems . The incorporation of recombinant L31 should be performed under physiologically relevant conditions (typically magnesium concentrations of 10-20 mM) to promote proper folding and association with the central protuberance of the 50S subunit where it normally resides . Success of reconstitution can be verified using structural techniques such as cryo-EM to confirm proper positioning at bridge B1b, with properly reconstituted samples showing clashscores comparable to those observed in native structures (approximately 4.84-15.11 as reported in previous studies) . Functional validation of the reconstituted ribosomes should include translation assays comparing activity with ribosomes containing native L31 protein, with particular attention to activities that might be affected by 30S-50S subunit interactions since L31 contributes to intersubunit bridge formation .

Structural Biology and Molecular Interactions

How does the binding of tigecycline to Acinetobacter baumannii 70S ribosomes affect the conformation and function of 50S ribosomal protein L31 type B?

Tigecycline binding to Acinetobacter baumannii 70S ribosomes induces significant conformational changes that propagate to the 50S ribosomal protein L31 type B. Structural studies have revealed that when tigecycline binds to its primary site at the interface of the 30S head and body, it appears to lock the 30S head to the 30S body, reducing head swivel motion and stabilizing the entire ribosomal structure . More surprisingly, additional tigecycline binding was observed at the central protuberance of the 50S subunit, where three tigecycline molecules bind in a cavity between the 23S rRNA, the 5S rRNA, and protein bL27 . These three molecules interact with one another through stacking interactions and through bridging magnesium ions, creating a complex network of interactions with the surrounding ribosomal components . The binding of tigecycline at this secondary site has long-range effects that extend to ribosomal protein L31, which forms part of bridge B1b connecting the ribosomal subunits . Specifically, the conformational shifts induced by tigecycline binding appear to make bL31 more rigid and better resolved in structural studies compared to when the ribosome is bound to amikacin, suggesting a potential functional impact on intersubunit dynamics during translation .

What are the differences in interaction patterns between 50S ribosomal protein L31 type B and various antibiotics targeting the bacterial ribosome?

The interaction patterns between 50S ribosomal protein L31 type B and various antibiotics reveal significant differences in binding mechanisms and conformational effects. When comparing tigecycline and amikacin, two clinically relevant antibiotics used against Acinetobacter baumannii, distinct patterns emerge in how they influence L31 dynamics . Tigecycline has been observed to induce increased rigidity in protein bL31, making it more well-resolved in structural studies compared to amikacin-bound ribosomes, where the protein appears less defined . This differential effect likely stems from tigecycline's ability to bind not only to its primary site at the interface of the 30S head and body but also to a secondary site at the central protuberance of the 50S subunit near where L31 is positioned . Principal component analysis of ribosome structures reveals that the largest variation in data for tigecycline-bound ribosomes is primarily due to 50S-30S intersubunit rotation, whereas amikacin-bound ribosomes show variations from both 30S head rotation and intersubunit rotation . These differences suggest that different classes of antibiotics may affect L31 function through distinct mechanisms: either through direct interactions with the protein and its surrounding environment or through allosteric effects that propagate through the ribosomal structure to impact L31's role in bridge B1b formation and stability .

What structural features of 50S ribosomal protein L31 type B contribute to its role in bridge B1b formation, and how might these be targeted in antibiotic development?

The 50S ribosomal protein L31 type B contains specific structural features that are crucial for its function in forming bridge B1b between the 30S and 50S ribosomal subunits. Although the complete atomic structure of L31 type B from Acinetobacter baumannii is not fully detailed in the provided research, structural studies of the 70S ribosome reveal that the protein resides at the central protuberance and contributes to intersubunit connections . The protein appears to work in concert with uL5, which is pulled away from its interaction with the 30S when tigecycline binds to the central protuberance, suggesting a coordinated mechanism of bridge formation . The high conservation of basic and polar amino acids in L31 (as seen in the sequence "MRKDIHPAYQQVLFHDTNADVYFLIGSTIQQTKQTKEYPGQVYPYVTLDISSASHPFYTGEVRQASNEGRVASFNKRFARFNRKS") likely facilitates interactions with negatively charged rRNA components across the subunit interface . For antibiotic development, these insights suggest potential strategies for targeting bridge B1b formation: compounds could be designed to either stabilize L31 in a conformation that prevents proper bridge formation or to disrupt specific interactions between L31 and its binding partners in the 30S subunit . The observation that tigecycline binding at the central protuberance affects L31 rigidity provides a proof-of-concept that small molecules can indeed modulate this protein's dynamics, potentially offering a novel mechanism for ribosome-targeting antibiotics distinct from those that directly inhibit peptidyl transferase activity or decoding .

Experimental Evolution and Resistance Mechanisms

How does long-term evolution of Acinetobacter baumannii under different nutritional conditions affect the expression and function of 50S ribosomal protein L31 type B?

Long-term experimental evolution of multidrug-resistant Acinetobacter baumannii under different nutritional conditions has revealed complex genomic changes that could potentially impact ribosomal proteins like L31 type B. When A. baumannii was cultured for 8000 generations under either starvation conditions (EAB1) or nutrient-rich conditions (EAB2), significant genomic alterations were observed, primarily mediated by insertion sequence (IS)-mediated insertions and deletions . Although specific changes to the rpmE2 gene (which encodes 50S ribosomal protein L31 type B) were not directly reported, the extensive genomic reorganization observed suggests potential impacts on ribosomal protein expression and function . The evolved strains exhibited increased virulence in mouse infection models, with distinct modes of action: EAB1 showed enhanced ability to cross epithelial barriers and evade immune responses, while EAB2 demonstrated stronger attachment to epithelial cells and triggered increased proinflammatory cytokine production . These phenotypic changes likely involve adaptations in cellular components including ribosomes, which are central to protein synthesis and cellular responses to environmental stress . The different evolutionary trajectories under contrasting nutritional conditions highlight the importance of considering environmental factors when studying ribosomal protein function and suggest that L31 type B expression or activity might be differentially regulated under resource-limited versus resource-abundant conditions .

What methodologies should be employed to investigate the role of 50S ribosomal protein L31 type B in antibiotic resistance mechanisms of Acinetobacter baumannii?

Investigating the role of 50S ribosomal protein L31 type B in antibiotic resistance mechanisms of Acinetobacter baumannii requires a multi-faceted methodological approach combining genetic, structural, and functional techniques. Researchers should begin with comparative genomics, analyzing the rpmE2 gene sequence across antibiotic-resistant and susceptible A. baumannii strains to identify potential mutations or polymorphisms associated with resistance phenotypes . CRISPR-Cas9 gene editing can be employed to generate precise deletions or mutations in the rpmE2 gene, followed by antibiotic susceptibility testing to directly assess the protein's contribution to resistance . High-resolution structural studies using cryo-electron microscopy would be essential for visualizing how L31 type B conformation changes in resistant strains when exposed to antibiotics, with particular attention to its position in bridge B1b and any alterations in ribosome dynamics . Ribosome profiling experiments comparing wild-type and L31-modified strains during antibiotic exposure could reveal changes in translation patterns that might contribute to resistance . Additionally, researchers should consider experimental evolution approaches similar to those used in previous studies, subjecting A. baumannii to long-term growth under antibiotic selective pressure while monitoring changes in the rpmE2 gene and L31 protein to identify adaptive mutations . Complementary proteomics analysis would help determine if L31 type B undergoes post-translational modifications in resistant strains that might affect its function or interaction with antibiotics like tigecycline that have been shown to influence its structural stability .

How can recombinant 50S ribosomal protein L31 type B be used to elucidate the binding mechanism of tigecycline at the secondary binding site in the 50S subunit?

Recombinant 50S ribosomal protein L31 type B can serve as a powerful tool for investigating tigecycline's secondary binding site at the central protuberance of the 50S ribosomal subunit through a series of targeted biochemical and structural approaches. Site-directed mutagenesis of the recombinant L31 protein should be performed to systematically alter amino acids potentially involved in the propagation of conformational changes induced by tigecycline binding, followed by incorporation of these variants into reconstituted ribosomes to assess effects on antibiotic binding affinity and ribosomal function . Researchers can conduct competitive binding assays using fluorescently labeled tigecycline and purified recombinant L31 in the context of partial ribosomal constructs to determine whether L31 directly contributes to the formation or stability of the secondary binding pocket that accommodates the three stacked tigecycline molecules observed in structural studies . Hydrogen-deuterium exchange mass spectrometry comparing dynamics of wild-type and modified L31 in the presence of tigecycline would reveal regions of the protein that become more protected or exposed upon antibiotic binding, providing insights into conformational changes . Cross-linking experiments between recombinant L31 and neighboring ribosomal components (particularly the 23S rRNA, 5S rRNA, and protein bL27) in the presence and absence of tigecycline would help map the exact network of interactions affected by antibiotic binding . Finally, cryo-EM analysis of ribosomes reconstituted with labeled recombinant L31 would allow direct visualization of how tigecycline binding at the central protuberance affects L31's position and rigidity, particularly in relation to bridge B1b formation and the large conformational change observed in U2308 of the 23S rRNA that flips out to interact with tigecycline's 9-t-butylglycylamido moiety .

Structural Data Interpretation

How should researchers interpret variations in model validation metrics for Acinetobacter baumannii 50S ribosomal components across different antibiotic-bound states?

Interpretation of model validation metrics for Acinetobacter baumannii 50S ribosomal components requires careful consideration of both technical and biological factors. When comparing metrics across different antibiotic-bound states, researchers should note the significant variations observed in previous studies: for instance, clashscores ranged from 4.84 in amikacin-ribosome 50S to 15.11 in tigecycline-ribosome 30S head, indicating substantial differences in atomic model quality across different ribosomal regions and drug-bound states . Correlation coefficients (CC) between model and map fit also showed meaningful variation, from 0.70 for tigecycline-ribosome 30S head to 0.86 for both amikacin-ribosome 50S and tigecycline-ribosome 50S, suggesting that certain regions may be better resolved or more stabilized in specific drug-bound states . Protein geometry parameters like Ramachandran favored percentages (ranging from 90.54% to 95.42%) and rotamer outliers (5.70% to 11.24%) further demonstrate how antibiotic binding can differentially affect local conformational stability . When these metrics vary significantly between conditions, researchers should consider whether such differences reflect genuine biological phenomena (such as drug-induced stabilization of certain regions) or technical limitations in data collection or processing . The observation that protein bL31 is more well-resolved in tigecycline-bound structures than in amikacin-bound ones suggests that such variations can indeed reflect biologically relevant changes in protein dynamics rather than merely technical artifacts .

What are the key challenges in accurately modeling 50S ribosomal protein L31 type B in cryo-EM structures, and what methodological approaches can address these challenges?

Accurately modeling 50S ribosomal protein L31 type B in cryo-EM structures presents several significant challenges that require sophisticated methodological solutions. The primary difficulty lies in L31's relatively small size (84 amino acids) and potential flexibility, as evidenced by its variable resolution in different antibiotic-bound states where it appears more well-resolved in tigecycline-bound structures compared to amikacin-bound ones . This flexibility seems biologically relevant to its function in bridge B1b formation between ribosomal subunits but creates modeling challenges . Additionally, L31's location at the central protuberance places it in a complex environment with numerous potential interactions with surrounding rRNA and proteins that must be accurately captured . To address these challenges, multibody refinement has proven effective by treating different ribosomal domains (50S, 30S body, and 30S head) as separate rigid bodies, allowing for better resolution of dynamic components like L31 . This approach has yielded maps with resolutions ranging from 2.5Å to 3.0Å for different ribosomal components . Principal component analysis can complement this method by identifying major modes of conformational variation that affect L31 positioning, such as the intersubunit rotation observed in tigecycline-bound ribosomes . For model building and validation, RNA geometry validation parameters (sugar pucker outliers and backbone conformation outliers) and protein geometry metrics (Ramachandran plots and rotamer analysis) should be carefully monitored, with previous studies reporting backbone conformation outlier percentages of 15.89-22.85% for ribosomal RNA components .

Practical Research Considerations

What quality control measures should be applied when working with recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B in functional studies?

Implementing rigorous quality control measures is essential when working with recombinant Acinetobacter baumannii 50S ribosomal protein L31 type B in functional studies to ensure reliable and reproducible results. Purity assessment should be conducted using SDS-PAGE to verify that the protein meets or exceeds the standard threshold of >85% purity, with additional mass spectrometry analysis recommended to confirm the exact molecular weight and sequence integrity of the 84-amino acid protein . Functional verification through binding assays with ribosomal RNA components, particularly those found at the central protuberance and those involved in bridge B1b formation, can confirm that the recombinant protein maintains its native interaction capabilities . Researchers should perform thermal stability assessments using differential scanning fluorimetry to ensure the protein maintains proper folding under experimental conditions, comparing results with established parameters for ribosomal proteins . Activity assays measuring the protein's ability to facilitate ribosomal subunit association would provide functional validation, with comparison to native ribosomes serving as positive controls . Given L31's role in antibiotic binding sites, particularly for tigecycline, drug-binding assays could serve as additional functional tests to confirm proper protein conformation . For long-term experiments, stability monitoring through regular SDS-PAGE analysis of stored aliquots is recommended, with adherence to optimal storage conditions (-20°C/-80°C for long-term storage, 4°C for up to one week for working aliquots) to prevent degradation that could compromise experimental outcomes .

How should researchers design experiments to investigate potential post-translational modifications of 50S ribosomal protein L31 type B in Acinetobacter baumannii under different stress conditions?

Designing experiments to investigate potential post-translational modifications (PTMs) of 50S ribosomal protein L31 type B under different stress conditions requires a comprehensive approach combining various analytical techniques. Researchers should first establish baseline PTM profiles of L31 from A. baumannii grown under standard conditions using high-resolution mass spectrometry approaches such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electron-transfer dissociation (ETD) or electron-capture dissociation (ECD) fragmentation methods, which are particularly effective for identifying modifications like phosphorylation, methylation, and acetylation . Parallel experiments should expose A. baumannii cultures to relevant stress conditions including antibiotic exposure (particularly tigecycline and amikacin, given their known interactions with ribosomes), nutrient limitation (similar to the EAB1 evolution conditions), and oxidative stress, followed by ribosome isolation and L31 purification using affinity chromatography or gel extraction methods . Two-dimensional gel electrophoresis comparing L31 protein spots across different conditions can provide initial evidence of PTMs that alter protein charge or mass . For site-specific identification of modifications, enrichment strategies specific to common PTMs (such as titanium dioxide for phosphopeptides or antibody-based enrichment for acetylated lysines) should be employed prior to MS analysis . To connect identified PTMs with functional consequences, researchers should generate recombinant L31 variants mimicking or lacking the identified modifications and assess their impact on ribosome assembly, stability, and response to antibiotics through reconstitution experiments and structural studies .

Therapeutic Potential and Drug Development

Based on structural data of Acinetobacter baumannii 70S ribosomes, how might 50S ribosomal protein L31 type B be exploited as a target for novel antibiotic development?

Structural data from Acinetobacter baumannii 70S ribosomes reveals several features of 50S ribosomal protein L31 type B that could be exploited for novel antibiotic development. The protein's critical role in bridge B1b formation between ribosomal subunits presents an opportunity to design compounds that disrupt this intersubunit connection, thereby interfering with ribosome function during translation . The observation that tigecycline binding at the central protuberance affects L31 conformation and rigidity suggests a mechanism for rational drug design: compounds could be developed to mimic this effect but with greater specificity for L31's structural environment . The binding pocket that accommodates three tigecycline molecules near L31, involving interactions with 23S rRNA, 5S rRNA, and protein bL27, provides a structurally defined target site for designing new molecules with optimized binding properties . Additionally, the long-range conformational effects observed when tigecycline binds at this site, including the significant movement of U2308 of the 23S rRNA and altered positioning of uL5, suggest that compounds targeting this region could have amplified impacts on ribosome dynamics beyond local interactions . Drug development strategies might focus on compounds that either stabilize L31 in non-functional conformations or prevent its proper incorporation into the ribosome, with the advantage that targeting bridge components might present a higher barrier to resistance development since mutations affecting these critical interfaces often come with significant fitness costs to the bacteria .

What experimental approaches are most suitable for assessing the specificity of compounds targeting the 50S ribosomal protein L31 type B region in Acinetobacter baumannii versus human ribosomes?

Assessing the specificity of compounds targeting the 50S ribosomal protein L31 type B region requires a multi-faceted experimental approach that carefully evaluates bacterial versus human ribosome interactions. Comparative structural analysis should be the starting point, utilizing cryo-electron microscopy to determine high-resolution structures of both A. baumannii 70S ribosomes and human 80S ribosomes in complex with candidate compounds, focusing particularly on the central protuberance region where L31 resides in bacterial ribosomes . In vitro translation assays using purified bacterial and mammalian translation systems can quantitatively assess inhibitory effects on protein synthesis, with IC50 determination providing a measure of selectivity between the two systems . Binding affinity studies utilizing surface plasmon resonance or isothermal titration calorimetry should compare the interaction kinetics and thermodynamics of compounds with isolated bacterial L31 protein versus its human counterparts . Researchers should implement photoaffinity labeling with compound analogs containing photoreactive groups to identify precise binding sites across both ribosome types, followed by mass spectrometry analysis to map cross-linked peptides . Mutagenesis experiments targeting specific residues in bacterial L31 that differ from human homologs can identify key specificity determinants, with testing of mutant ribosomes against candidate compounds to validate their role in selective binding . Computational approaches including molecular dynamics simulations comparing the binding energy landscapes across bacterial and human ribosomes can further guide optimization of compound selectivity, with particular attention to the unique structural features of the binding pocket near 23S rRNA and 5S rRNA in the bacterial ribosome that accommodated three tigecycline molecules in previous structural studies .