Recombinant Staphylococcus aureus Elongation factor G (fusA), partial

What is the primary function of Elongation Factor G (fusA) in Staphylococcus aureus?

Elongation Factor G (EF-G), encoded by the fusA gene in S. aureus, is crucial in two key steps of bacterial protein synthesis: elongation and ribosome recycling. During elongation, EF-G catalyzes the translocation of peptidyl-tRNA from the A-site to the P-site on the ribosome following peptide bond formation. In ribosome recycling, EF-G works with ribosome recycling factor (RRF) to split the 70S ribosome into subunits, allowing them to participate in new rounds of translation . Both functions require GTP hydrolysis, with EF-G undergoing conformational changes between GTP and GDP states that are essential for its proper function .

How does fusidic acid inhibit S. aureus protein synthesis?

Fusidic acid works by blocking Elongation Factor G on the ribosome after GTP hydrolysis. The antibiotic traps EF-G in an intermediate conformation between the GTP and GDP states, preventing its release from the post-translocation complex . Specifically, fusidic acid binds to a hydrophobic pocket formed between domains I, II, and III of EF-G and interacts directly with key residues like Phe-88 (equivalent to Phe-90 in T. thermophilus) . This interaction prevents the switch II region from adopting its GDP conformation while binding only after switch I has left its ordered GTP conformation, effectively locking EF-G in this intermediate state and inhibiting both elongation and ribosome recycling steps of bacterial protein synthesis .

What are the key structural features of S. aureus EF-G?

The crystal structure of S. aureus EF-G has been solved to 1.9 Å resolution, revealing several key structural features :

What are the common fusA mutations associated with fusidic acid resistance?

Multiple fusA mutations have been identified in fusidic acid-resistant S. aureus isolates. These include:

• F88L mutation: One of the primary mutations found in clinical isolates that leads to strong fusidic acid resistance

• M16I/M16V mutations: Often found as secondary mutations that compensate for fitness loss caused by F88L while maintaining resistance

• P406L, H457Y, and L461K mutations: Confirmed to confer fusidic acid resistance when introduced into susceptible S. aureus strains

Sequence analysis has revealed at least 14 different amino acid exchanges restricted to 13 amino acid residues within EF-G in clinical isolates and laboratory-derived resistant mutants . Many of these resistance mutations are located at domain interfaces of EF-G, particularly around the hydrophobic pocket between domains I, II, and III where fusidic acid binds .

How do fusA mutations affect EF-G function and bacterial fitness?

Primary resistance mutations like F88L significantly impact EF-G function and bacterial fitness in several ways:

• Slower translocation: The F88L mutation restricts the conformational changes of EF-G, leading to reduced efficiency in tRNA-mRNA translocation

• Reduced ribosome recycling: The mutation impairs the ability of EF-G to participate effectively in ribosome recycling

• Increased tRNA drop-off: Resistance mutations can increase the rate of tRNA dissociation from the ribosome during translation

• Growth defects: These functional impairments manifest as significant fitness costs, with F88L mutants showing notable growth defects in S. aureus

The restricted conformational dynamics caused by resistance mutations appears to be the primary mechanism underlying these functional deficits, highlighting the critical importance of EF-G's conformational flexibility for proper function .

What is the mechanism of fitness compensation in fusidic acid-resistant strains?

Secondary compensatory mutations, such as M16I when paired with F88L, can restore fitness while maintaining resistance to fusidic acid . This fitness compensation occurs through:

• Restoration of conformational dynamics: Compensatory mutations appear to restore the ability of EF-G to undergo the necessary conformational changes for efficient function while maintaining an altered fusidic acid-binding pocket

• Improved translation efficiency: The double mutant (F88L/M16I) shows improved translation rates compared to the single F88L mutant

• Reduced tRNA drop-off: Compensatory mutations decrease the rate of premature tRNA dissociation during translation

• Enhanced ribosome recycling: The secondary mutations help restore efficient ribosome recycling capability

What are the optimal methods for purifying recombinant S. aureus EF-G?

Based on research protocols, recombinant S. aureus EF-G can be optimally purified using the following approach:

• Expression system selection:

  • E. coli BL21(DE3) or similar expression strains

  • Expression vector containing the S. aureus fusA gene with an N-terminal His6-tag

  • IPTG-inducible promoter system

• Purification protocol:

  • Affinity chromatography using Ni-NTA resin for initial capture

  • Intermediate purification using ion exchange chromatography

  • Final polishing with size exclusion chromatography

• Buffer optimization:

  • Inclusion of 5-10 mM MgCl₂ in all buffers (critical for GTPase stability)

  • Addition of 10% glycerol to prevent protein aggregation

  • Maintenance of reducing conditions with DTT or β-mercaptoethanol

This multi-step purification approach yields highly pure and active recombinant S. aureus EF-G suitable for functional and structural studies .

What in vitro assays can be used to evaluate S. aureus EF-G function?

Several complementary assays can be used to evaluate the function of recombinant S. aureus EF-G:

• GTPase activity assay:

  • Measures intrinsic or ribosome-stimulated GTP hydrolysis rates

  • Can use radiolabeled [³H]GTP or colorimetric methods

  • Useful for comparing wild-type and mutant EF-G variants

• Ribosomal complex formation assay:

  • Uses [³H]GTP to quantify stable EF-G-ribosome complexes on nitrocellulose filters

  • Can detect differences in fusidic acid binding between EF-G variants

  • Example: F88L and F88L/M16I mutants show greatly reduced complex formation with fusidic acid (5-8%) compared to wild-type (90%)

• Translocation assay:

  • Measures the ability of EF-G to catalyze tRNA-mRNA movement on the ribosome

  • Can be monitored using fluorescence methods or biochemical approaches

  • Key for understanding how mutations affect the core function of EF-G

• Ribosome recycling assay:

  • Evaluates EF-G efficiency in ribosome splitting in conjunction with RRF

  • Important for assessing the second major function of EF-G

These assays provide a comprehensive functional profile of wild-type and mutant EF-G variants, enabling detailed mechanistic studies of antibiotic resistance and fitness compensation .

How can site-directed mutagenesis be used to study specific residues in S. aureus EF-G?

Site-directed mutagenesis is a powerful approach for studying specific residues in S. aureus EF-G:

• Technical approach:

  • PCR-based methods using complementary primers containing the desired mutation

  • Template digestion with DpnI to remove parental DNA

  • Transformation into competent E. coli cells followed by sequencing verification

• Successful applications:

  • Introduction of resistance mutations (F88L, P406L, H457Y, L461K) into wild-type fusA

  • Creation of compensatory mutations (M16I) in resistant backgrounds

  • Confirmation of the role of specific mutations in fusidic acid resistance

• Functional validation:

  • Expression of mutant fusA alleles on plasmids in susceptible S. aureus strain RN4220

  • Determination of minimal inhibitory concentrations (MICs) for fusidic acid

  • Correlation of introduced mutations with resistance phenotypes

Research has demonstrated that introduction of mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y, and L461K into susceptible S. aureus confers fusidic acid resistance comparable to clinical isolates with the same mutations, confirming the direct role of these specific amino acid substitutions in resistance .

How can structural information about S. aureus EF-G guide novel antibiotic development?

The high-resolution (1.9 Å) crystal structure of S. aureus EF-G provides valuable information for rational antibiotic design:

• Target site identification:

  • The unique conformation of S. aureus EF-G compared to other bacterial species offers opportunities for selective inhibition

  • Differences in the switch I and switch II regions could be exploited for developing S. aureus-specific inhibitors

  • The 25 Å displacement of domain IV relative to domain G presents unique binding sites not found in other bacteria

• Resistance circumvention:

  • Understanding how mutations like F88L confer resistance can guide the design of new drugs that maintain activity against resistant strains

  • Identification of conserved regions less prone to resistance-conferring mutations

  • Development of compounds that bind to EF-G through different interactions than fusidic acid

• Structure-based approaches:

  • Virtual screening against the S. aureus EF-G structure

  • Fragment-based drug discovery targeting critical functional regions

  • Rational modification of existing translation inhibitors based on binding pocket analysis

The species-specific structural features of S. aureus EF-G provide promising opportunities for the development of narrow-spectrum antibiotics with potentially reduced resistance emergence .

What is the relationship between EF-G conformational dynamics and antibiotic resistance?

Research has revealed critical insights into the relationship between EF-G conformational dynamics and antibiotic resistance:

The F88L mutation is particularly interesting as it affects a residue (Phe-88) thought to be important for transmitting conformational changes between the GTP and GDP forms of EF-G. This provides direct evidence for the crucial role of conformational dynamics in both EF-G function and antibiotic resistance .

How does the study of S. aureus EF-G contribute to our understanding of bacterial translation?

The study of S. aureus EF-G has made significant contributions to our understanding of bacterial translation:

• Conformational dynamics:

  • The dramatically different conformation of S. aureus EF-G compared to other bacterial EF-Gs highlights the dynamic nature of this protein

  • The functional consequences of restricted dynamics (in F88L mutants) demonstrate how conformational changes are coupled to translation efficiency

• Energy transduction mechanisms:

  • Research on EF-G mutations reveals how GTP hydrolysis is coupled to mechanical work (translocation)

  • The importance of specific residues like Phe-88 in this energy coupling process has been identified

• Species-specific adaptations:

  • The unique conformation of S. aureus EF-G suggests species-specific adaptations in translation machinery

  • These differences may reflect adaptations to different growth conditions or selective pressures

• Antibiotic mechanisms:

  • Detailed understanding of how fusidic acid traps EF-G in an intermediate conformation between GTP and GDP states

  • Insight into how this trapping disrupts the translation cycle

This research exemplifies how studies motivated by clinical concerns (antibiotic resistance) simultaneously advance our understanding of fundamental biological processes (translation) and provide practical benefits (new drug development strategies).

What are promising approaches for developing new inhibitors of S. aureus EF-G?

Several promising approaches for developing new S. aureus EF-G inhibitors include:

• Structure-guided modification of fusidic acid:

  • Creating derivatives that maintain activity against resistant variants

  • Modifying fusidic acid to interact with conserved regions less prone to mutations

  • Developing analogs with improved pharmacokinetic properties

• Targeting novel binding sites:

  • Exploiting the unique conformation of S. aureus EF-G to identify binding pockets distinct from the fusidic acid binding site

  • Developing allosteric inhibitors that affect EF-G function through different mechanisms

  • Targeting the interfaces between EF-G domains that are critical for conformational changes

• Combination approaches:

  • Designing dual-action inhibitors that target both EF-G and other components of the translational machinery

  • Developing compounds that synergize with existing antibiotics

• Rational design based on resistance mechanisms:

  • Using insights from fitness-compensated resistant strains to develop inhibitors that target both wild-type and resistant EF-G variants

  • Focusing on molecular features that cannot be easily modified without severe fitness costs

How might genomic surveillance of fusA mutations inform clinical treatment decisions?

Genomic surveillance of fusA mutations could inform clinical treatment decisions in several ways:

• Resistance prediction:

  • Rapid identification of known resistance mutations (F88L, P406L, H457Y, L461K, etc.) to predict fusidic acid treatment outcomes

  • Detection of compensatory mutations (M16I) that indicate stable resistant strains

• Treatment guidance:

  • Informing appropriate antibiotic selection early in treatment

  • Avoiding fusidic acid monotherapy in infections with high prevalence of resistance mutations

  • Guiding combination therapy approaches

• Resistance monitoring:

  • Tracking the emergence and spread of specific fusA mutations in healthcare settings

  • Informing infection control measures and antibiotic stewardship programs

• Novel mutation detection:

  • Identifying previously unreported fusA mutations

  • Correlating these with treatment outcomes to expand our understanding of resistance mechanisms

The detailed molecular understanding of how specific fusA mutations affect fusidic acid binding and EF-G function provides a strong foundation for developing such surveillance approaches .

What techniques might provide deeper insights into the dynamic conformational changes of EF-G during translation?

Advanced techniques that could provide deeper insights into EF-G conformational dynamics include:

• Time-resolved cryo-electron microscopy:

  • Capturing EF-G in different conformational states during the translation cycle

  • Visualizing transient intermediates not accessible by traditional structural methods

• Single-molecule FRET studies:

  • Monitoring real-time conformational changes in EF-G during interaction with the ribosome

  • Comparing dynamics of wild-type and mutant EF-G variants

• Hydrogen-deuterium exchange mass spectrometry:

  • Probing conformational flexibility and solvent accessibility changes

  • Identifying regions with altered dynamics in resistant and compensated EF-G variants

• Molecular dynamics simulations:

  • Predicting conformational transitions between different functional states

  • Assessing how specific mutations alter the energy landscape of EF-G conformations

• NMR spectroscopy:

  • Investigating solution dynamics of EF-G domains

  • Characterizing transient interactions between domains

These techniques could significantly enhance our understanding of how EF-G conformational dynamics contribute to function and how mutations alter these dynamics to confer resistance while maintaining or restoring fitness .