Recombinant Lactobacillus plantarum Glycine--tRNA ligase beta subunit (glyS), partial

What is the structural organization of the Glycine-tRNA ligase (GlyRS) in Lactobacillus plantarum?

The GlyRS in L. plantarum exhibits a heterotetrameric α₂β₂ structure that is found in most eubacteria, distinguishing it from the dimeric α₂ structure present in archaea, eukaryotes, and some eubacteria. The crystal structure reveals that the α₂β₂ GlyRS from L. plantarum (LpGlyRS) adopts a distinctive X-shaped architecture, with the full-length α and β subunits arranged in a specific configuration. This X-shaped structure is homologous to that observed in Escherichia coli GlyRS (EcGlyRS) .

The β subunit of LpGlyRS contains five domains with specialized functions:

• The N-terminal domains facilitate the formation of the active pocket by the α-subunit

• The C-terminal domains contribute to tRNA binding, showing significant structural similarity to tRNA CCA-adding enzymes and a tRNA recognition domain in alanyl-tRNA synthetase (AlaRS)

This unique structural arrangement enables the enzyme to effectively recognize and aminoacylate tRNA^Gly, with specific domains of the α and β subunits interacting with different regions of the L-shaped tRNA structure .

How does the β subunit of L. plantarum GlyRS interact with tRNA^Gly?

The interaction between LpGlyRS β subunit and tRNA^Gly involves multiple specific contacts that ensure accurate recognition and aminoacylation. Based on tRNA docking models and biochemical studies:

• The α and β subunits together interact with the 3'-end and the acceptor region of tRNA^Gly

• The C-terminal domain of the β subunit specifically interacts with the anticodon region of tRNA^Gly

• The loop between helices H18 and H19 (Loop H18-19) and the N-terminal part of H19 from the HD domain insert into the major groove of the tRNA^Gly acceptor stem

• These domains form base-specific hydrogen-bonding interactions with the first four base pairs (G1- C72, C2- G71, G3- C70, and G4- C69) of the acceptor stem

This recognition mechanism is critical for the accurate attachment of glycine to its cognate tRNA, ensuring fidelity in protein translation.

What are the optimal expression systems for producing recombinant L. plantarum GlyS β subunit?

For successful expression of recombinant L. plantarum GlyS β subunit, researchers should consider the following methodological approach:

• Vector Selection: An E. coli-Lactobacillus shuttle expression vector system provides optimal results. Current research demonstrates success using vectors with antibiotic-free screening markers such as the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene .

• Host Systems:

  • For initial cloning: Use asd gene-deficient E. coli (E. coli χ6212) as the plasmid donor

  • For expression: Use alr gene deletion L. plantarum strain NC8Δ as the host strain

• Expression Strategies: The pWCF expression vector has shown effectiveness for achieving expression of the target protein, using pgsA' gene product as an attachment matrix for the recombinant protein on the surface of L. plantarum .

This approach avoids the environmental impact of resistance screening markers while maintaining efficient expression.

What purification challenges are specific to the GlyS β subunit, and how can they be addressed?

Purification of the GlyS β subunit presents several challenges due to its structural properties and interaction tendencies:

• Subunit Dissociation: The α and β subunits tend to dissociate easily. This challenge can be addressed by:

  • Including tRNA binding during purification, as it results in a larger interface between the HD domain and the α subunit (608 Ų versus 252 Ų)

  • Utilizing buffer conditions that stabilize the heterotetrameric structure

• Domain Flexibility: The B2 domain of the β subunit shows weak electron density in crystallographic studies due to its dynamic nature . Strategies to address this include:

  • Using chemical crosslinking to stabilize the conformation

  • Employing size exclusion chromatography under conditions that promote complex integrity

• Maintaining Native Conformation: The unique X-shaped architecture requires careful handling during purification. Methods include:

  • Avoiding harsh elution conditions that might disrupt the quaternary structure

  • Including glycerol (10-15%) in purification buffers to stabilize the protein conformation

These purification strategies help maintain the functional integrity of the complex enzyme for subsequent biochemical and structural studies.

What experimental approaches can determine the tRNA recognition elements specific to L. plantarum GlyRS?

To determine the tRNA recognition elements specific to L. plantarum GlyRS, researchers should employ a multi-faceted approach:

• Variant tRNA Analysis: Create systemic mutations in tRNA^Gly and test glycylation efficiency. Biochemical analysis using tRNA variants has identified several key determinants:

  • Previously known determinants: G1C72 and C2G71 base pairs, C35, C36, and U73

  • Newly identified elements: positions 4 and 69, with a preference for a purine base at position 4 and a pyrimidine base at position 69

• Acceptor Stem Mutation Studies: Replace elements of the tRNA^Gly acceptor stem with those from other tRNAs (e.g., tRNA^Ile) and measure aminoacylation activity:

• Chimeric tRNA Approach: Substitute individual structural elements (D, anticodon, T stem sequences) of tRNA^Gly with those of tRNA^Ile to isolate recognition determinants .

• Structural Analysis: Use crystallography or cryo-EM combined with molecular modeling to visualize the interaction interfaces.

These methodologies collectively provide a comprehensive understanding of the specific nucleotide requirements for efficient aminoacylation by LpGlyRS.

How does the aminoacylation mechanism of L. plantarum GlyRS differ from other bacterial GlyRS enzymes?

The aminoacylation mechanism of L. plantarum GlyRS presents several distinctive features compared to other bacterial GlyRS enzymes:

• Recognition of tRNA Base Pairs 4-69: Unlike E. coli GlyRS, LpGlyRS shows a strong preference for a purine base at position 4 and a pyrimidine base at position 69 in tRNA^Gly. This combination enhances glycylation efficiency significantly .

• Structural Rearrangements During tRNA Binding: Upon tRNA binding, LpGlyRS undergoes a large conformational change:

  • The C-terminal part of the β subunit (HD and ABD domains) rotates approximately 30°

  • This rotation mainly occurs at the linker between the B3 and HD domains

  • The ABD moves inward about 35 Å to make contact with the anticodon loop of tRNA^Gly

• Mini-helix Recognition: Unlike E. coli GlyRS that can glycylate a mini-helix RNA consisting of the acceptor and T-stems of tRNA^Gly, LpGlyRS requires the complete L-shaped tRNA structure for efficient aminoacylation .

• Interface Stabilization: tRNA binding significantly increases the interface between the HD domain and the α subunit (608 Ų versus 252 Ų), suggesting that tRNA binding stabilizes the heterotetrameric complex in LpGlyRS more substantially than in other bacterial GlyRS enzymes .

These mechanistic differences reflect evolutionary adaptations that may relate to the specific cellular environment and translation requirements of L. plantarum.

What potential advantages does the L. plantarum expression system offer for therapeutic protein delivery compared to other bacterial systems?

The L. plantarum expression system offers several distinct advantages for therapeutic protein delivery:

• Safety Profile:

  • Lacks lipopolysaccharides (LPS) in cell walls, unlike Gram-negative bacteria such as E. coli

  • Can be administered orally without risk of endotoxic shock

  • Internationally recognized as a food-grade microorganism with GRAS (Generally Recognized As Safe) status

• Gastrointestinal Survival:

  • Can survive gastrointestinal passage and colonize the gastrointestinal tract

  • Provides continuous in situ delivery of peptides or proteins

  • Reduces exposure of therapeutic proteins to gastric acid, bile, and digestive enzymes

• Immune Response Advantages:

  • Demonstrated ability to activate dendritic cells in Peyer's patches

  • Increases CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes

  • Enhances B220+IgA+ cells in Peyer's patches and IgA production in the intestine and lungs

• Expression System Versatility:

  • Supports both secreted and cell wall-anchored expression of recombinant proteins

  • Can incorporate intestinal trypsin sites for controlled release of therapeutic peptides

These advantages make L. plantarum particularly suitable for oral delivery of therapeutic proteins targeting intestinal disorders, infectious diseases, and systemic conditions requiring mucosal immune responses.

How can the GlyS β subunit knowledge contribute to developing novel biotechnological applications?

Understanding the GlyS β subunit structure and function opens several avenues for biotechnological applications:

• Enhanced Protein Expression Systems:

  • The unique domain architecture of the GlyS β subunit can inform the design of novel protein fusion tags

  • The tRNA recognition mechanisms can be exploited to develop systems with enhanced translational efficiency and accuracy

  • The X-shaped architecture provides insights for designing protein complexes with controlled spatial arrangement

• Targeted Drug Delivery:

  • The specific interaction between GlyS and tRNA can inspire the design of molecules that recognize specific RNA structures

  • This knowledge can be applied to developing targeted drug delivery systems for RNA-based therapeutics

  • Understanding the conformational changes upon tRNA binding offers insights for designing allosteric regulators

• Antimicrobial Development:

  • The structural differences between bacterial (α₂β₂) and human (α₂) GlyRS enzymes provide targets for selective antimicrobial agents

  • Compounds targeting the unique interface between α and β subunits or the specific tRNA recognition elements of bacterial GlyRS could serve as novel antibiotics

• Synthetic Biology Applications:

  • The aminoacylation specificity determinants can be engineered to incorporate non-canonical amino acids into proteins

  • This enables the production of proteins with novel functionalities for research and therapeutic applications

These applications represent promising directions for translating fundamental knowledge about GlyS structure and function into biotechnological innovations.

What are the optimal crystallization conditions for obtaining high-resolution structures of the L. plantarum GlyRS-tRNA complex?

Crystallization of the LpGlyRS-tRNA complex requires careful optimization of multiple parameters:

• Sample Preparation:

  • Purify LpGlyRS to >95% homogeneity using immobilized metal affinity chromatography followed by size exclusion chromatography

  • Prepare in vitro transcribed tRNA^Gly with homogeneous 3' CCA end

  • Form the complex with 1:1.2 molar ratio (protein:tRNA) in the presence of non-hydrolyzable ATP analogs and glycine or glycyl-adenylate analogs

• Crystallization Conditions:

  • Buffer composition: 100 mM Tris-HCl (pH 7.5-8.0), 10-15% PEG 3350, 100-200 mM MgCl₂

  • Additives: 5-10 mM spermidine, 1-5 mM spermine, 50-100 mM KCl

  • Temperature: 18-20°C using hanging or sitting drop vapor diffusion method

  • Drop ratio: 1:1 protein:reservoir with final drop size of 2-4 μL

• Cryoprotection Optimization:

  • Gradual transfer to mother liquor supplemented with 15-20% glycerol or ethylene glycol

  • Alternative: 25-30% PEG 400 or a mixture of low molecular weight PEGs

  • Flash-cooling in liquid nitrogen with minimal crystal handling

• Co-crystallization Strategies:

  • Include non-hydrolyzable ATP analogs (e.g., AMPCPP) and glycine

  • Pre-form the ternary complex (GlyRS- glycyl-adenylate- tRNA^Gly) before crystallization

  • Consider using tRNA variants with enhanced stability or crystallization properties

These optimized conditions have yielded crystals diffracting to resolutions of 2.6-3.0 Å, suitable for detailed structural analysis of the complex.

What advanced biophysical techniques can reveal the dynamic interactions between the GlyS β subunit and tRNA?

Several advanced biophysical techniques can provide insights into the dynamic interactions between GlyS β subunit and tRNA:

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET):

  • Label GlyS β subunit and tRNA^Gly with appropriate fluorophore pairs

  • Monitor real-time conformational changes during complex formation and aminoacylation

  • Quantify the dynamics of domain movements, particularly the 30° rotation observed in structural studies

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map regions of GlyS β subunit that undergo conformational changes upon tRNA binding

  • Identify protected regions that form direct contacts with tRNA

  • Determine the kinetics of structural rearrangements in different functional states

• Cryo-Electron Microscopy (Cryo-EM):

  • Visualize different conformational states of the GlyRS-tRNA complex

  • Capture transient intermediates in the aminoacylation reaction pathway

  • Resolve the flexibility of domains like B2 that show weak electron density in crystal structures

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Characterize domain dynamics and local conformational changes

  • Analyze chemical shift perturbations upon tRNA binding to map interaction interfaces

  • Study the dynamics of specific domains, such as the flexible B2 domain

• Molecular Dynamics (MD) Simulations:

  • Model the conformational changes of GlyS upon tRNA binding

  • Simulate the rotation of the HD and ABD domains

  • Predict the energetics of domain movements and identify key residues involved in the conformational transitions

These techniques, when used in combination, provide a comprehensive understanding of the dynamic processes involved in tRNA recognition and aminoacylation by LpGlyRS.

How can we address the challenges in increasing expression levels of the GlyS β subunit in recombinant L. plantarum systems?

Increasing expression levels of the GlyS β subunit in recombinant L. plantarum systems presents several challenges that can be addressed through systematic optimization:

• Codon Optimization Strategies:

  • Analyze the codon usage bias in L. plantarum and optimize the GlyS β subunit gene accordingly

  • Consider selective codon optimization of regions containing rare codons rather than whole-gene optimization

  • Implement a codon harmonization approach that mimics the translational rhythm of the native host

• Promoter and Regulatory Element Optimization:

  • Test a panel of constitutive and inducible promoters with varying strengths

  • Optimize the ribosome binding site (RBS) sequence and spacing

  • Incorporate transcription terminator sequences to prevent read-through transcription

• Secretion Signal Optimization:

  • Evaluate different signal peptides for optimal secretion or surface display

  • Consider the usp45 signal peptide from L. lactis or signals native to L. plantarum

  • Test fusion partners like polyglutamate synthase A (pgsA) for efficient surface display

• Culture Condition Optimization:

  • Systematic optimization of temperature, pH, and media composition

  • Implement fed-batch or continuous cultivation strategies

  • Evaluate the impact of growth phase on expression levels

• Genetic Stability Enhancement:

  • Develop chromosomal integration approaches for stable expression

  • Use antibiotic-free selection systems based on complementation of essential genes like asd and alr

  • Implement genetic circuits that couple expression to cellular growth

By integrating these approaches, researchers can overcome the expression limitations observed in current systems, where "5×GLP-1 expression did not provide an additional anti-diabetic effect, possibly due to the low levels produced" .

What are the unresolved questions regarding the evolutionary relationship between heterotetrameric and dimeric GlyRS enzymes?

Several unresolved questions remain regarding the evolutionary relationship between heterotetrameric (α₂β₂) and dimeric (α₂) GlyRS enzymes:

• Evolutionary Origin and Divergence:

  • When did the divergence between heterotetrameric and dimeric GlyRS occur in evolutionary history?

  • What selective pressures drove the maintenance of these distinct architectures in different lineages?

  • Did the heterotetrameric form evolve from a dimeric ancestor, or vice versa?

• Functional Equivalence vs. Specialization:

  • Do the structural differences between the two forms reflect functional specialization for different cellular environments?

  • Are there differences in aminoacylation efficiency, accuracy, or regulation between the two forms?

  • How do the different architectures affect interactions with other components of the translation machinery?

• Domain Fusion and Reorganization:

  • What was the evolutionary pathway that led to the fusion of α and β subunits in some bacterial lineages?

  • How did the unique domain architecture of the β subunit evolve, particularly the domains that show structural similarity to other enzymes like tRNA CCA-adding enzymes?

• Mechanistic Conservation:

  • Despite the structural differences, what mechanistic features are conserved between heterotetrameric and dimeric GlyRS enzymes?

  • How do the two types of enzymes achieve specificity for tRNA^Gly recognition?

  • Are there convergent solutions to the problem of specific glycylation?

Addressing these questions requires comparative genomic, structural, and biochemical studies across diverse organisms, potentially revealing fundamental principles of molecular evolution and enzyme function optimization.

How can insights from L. plantarum GlyS β subunit research be integrated with broader studies on Lactobacillus as a delivery system for therapeutic proteins?

The integration of L. plantarum GlyS β subunit research with studies on Lactobacillus as a therapeutic protein delivery system offers promising synergistic opportunities:

• Enhanced Expression Systems:

  • Knowledge of GlyRS structure and function can inform the design of optimized translation systems within L. plantarum

  • Understanding tRNA recognition elements can lead to improved codon optimization strategies for heterologous protein expression

  • The natural aminoacylation process can be engineered to incorporate non-canonical amino acids into therapeutic proteins

• Multi-functional Therapeutic Approaches:

  • The intrinsic anti-diabetic effect of L. plantarum observed in studies can be combined with the expression of therapeutic proteins for synergistic effects

  • This approach could address multiple aspects of complex diseases simultaneously, as suggested by findings that "lactobacilli themselves might be used as an alternative treatment method for type 2 diabetes"

• Strategic Protein Engineering:

  • The pentameric design approach used in GLP-1 studies can be applied to other therapeutic proteins

  • Incorporating intestinal trypsin sites within recombinant proteins can enable controlled release of active monomeric forms in the gut

  • Surface display versus secretion strategies can be optimized based on therapeutic requirements

• Immune Modulation Integration:

  • The demonstrated ability of recombinant L. plantarum to induce both systemic and mucosal immune responses can be harnessed for vaccine development

  • The activation of dendritic cells in Peyer's patches and increased numbers of CD4+ and CD8+ T cells can enhance the efficacy of therapeutic proteins targeting immune-related disorders

This integrated approach represents a promising direction for developing next-generation biotherapeutics that combine the beneficial properties of the delivery vehicle with the specific activity of the therapeutic protein.

What interdisciplinary approaches could advance our understanding of the structure-function relationship in GlyS and its potential applications?

Advancing our understanding of GlyS structure-function relationships and applications requires interdisciplinary approaches that bridge multiple scientific domains:

• Computational Biology and Structural Bioinformatics:

  • Apply machine learning algorithms to predict tRNA recognition determinants across different species

  • Use molecular dynamics simulations to explore the conformational landscape of GlyRS-tRNA interactions

  • Develop structure-based design of GlyRS variants with altered specificity or enhanced activity

• Synthetic Biology and Protein Engineering:

  • Create chimeric synthetases combining domains from different aaRSs to develop novel catalytic functions

  • Engineer the tRNA recognition mechanism for incorporation of non-canonical amino acids

  • Develop synthetic genetic circuits that utilize GlyRS-tRNA interactions as regulatory components

• Systems Biology and Metabolomics:

  • Investigate the impact of GlyRS activity on the global metabolic network of L. plantarum

  • Study how translation efficiency of glycine-rich proteins affects cellular physiology

  • Examine potential moonlighting functions of GlyRS beyond aminoacylation

• Immunology and Mucosal Biology:

  • Explore how recombinant L. plantarum expressing GlyS fragments or fusion proteins interacts with the immune system

  • Investigate potential adjuvant properties of GlyS domains for mucosal vaccine development

  • Study the interaction between L. plantarum and the host gastrointestinal environment

• Translational Research and Therapeutic Development:

  • Develop GlyS-based biosensors for detecting tRNA modifications or other RNA structures

  • Create screening platforms for identifying inhibitors of bacterial GlyRS as potential antibiotics

  • Design novel drug delivery systems based on the tRNA recognition mechanism

By fostering collaboration across these disciplines, researchers can accelerate the translation of fundamental insights into practical applications while deepening our understanding of this fascinating molecular machine.