Recombinant Mycoplasma pneumoniae UPF0134 protein MPN_010 (MPN_010)

What is the structural composition of Mycoplasma pneumoniae UPF0134 protein MPN_010?

MPN_010 contains a DUF16 (Domain of Unknown Function 16) domain with a distinctive structural arrangement. The crystal structure, determined at 1.8Å resolution, reveals that MPN_010 forms a homotrimer composed of two separated homotrimeric coiled-coils . The protein consists of 131 amino acid residues and has a UniProtKB accession number of P75103 . The shorter coiled-coil contains 11 highly conserved residues, while the longer coiled-coil comprises approximately nine heptad repeats with noncanonical sequences that induce a right-handed coiled-coil structure . This structural composition distinguishes MPN_010 from other known homotrimeric coiled-coil proteins.

What are the unique features of MPN_010's coiled-coil structures?

The longer coiled-coil structure in MPN_010 contains three distinguishable regions that confer unique structural properties compared to other known homotrimeric coiled-coils . The first part contains one stutter and is characterized by an unusual phenylalanine-rich region not found in other coiled-coil structures . The second part features a highly conserved glutamine-rich region, which is frequently observed in other trimeric coiled-coil structures . The third part consists of prototype heptad repeats that follow conventional coiled-coil arrangements . These distinct features suggest specialized functions and interactions for the MPN_010 protein.

What are the potential functions of MPN_010 based on structural analysis?

While the specific molecular function of MPN_010 remains officially unassigned in database annotations , structural analysis provides valuable insights into its potential roles. The presence of conserved coiled-coil domains suggests possible involvement in protein-protein interactions, structural maintenance, or cellular organization. Phylogenetic analysis has shown that the DUF16 family can be classified into five subclasses according to N-terminal sequences , indicating potential functional diversification across bacterial species. The unique phenylalanine-rich and glutamine-rich regions in the coiled-coil domains may mediate specific molecular interactions relevant to Mycoplasma pneumoniae biology. Based on structural comparisons with other coiled-coil proteins, researchers have speculated about potential roles in cellular adhesion, signaling, or maintaining bacterial cell architecture.

How might MPN_010 contribute to Mycoplasma pneumoniae pathogenesis?

While direct evidence linking MPN_010 to Mycoplasma pneumoniae pathogenesis is limited in current literature, several hypotheses can be formulated based on known mechanisms of MP infection. MP is a common respiratory pathogen affecting children and the elderly , with infection mechanisms involving adhesion to respiratory epithelial cells. The major adhesion factors in MP include the P1 and P30 proteins , which have been studied for vaccine development. MPN_010, with its coiled-coil structure, might participate in protein complexes involved in host-pathogen interactions or bacterial structural integrity during infection.

Immune responses to MP infection include both cellular and humoral components, with altered levels of CD3+ T cells, IgG, and inflammatory markers observed in infected patients . If MPN_010 is expressed during infection and exposed to the host immune system, it could potentially contribute to these immunological responses. This hypothesis warrants investigation through studies examining MPN_010 expression during different stages of infection and testing antibody responses to recombinant MPN_010 in patients with MP pneumonia.

What experimental approaches can be used to determine the biological function of MPN_010?

A comprehensive approach combining these methodologies would provide complementary data to elucidate MPN_010's function. Gene knockout studies would reveal whether MPN_010 is essential for MP viability or contributes to specific phenotypes such as growth rate, morphology, or infectivity. Protein-protein interaction studies would identify molecular partners that could indicate involvement in specific cellular processes. Structural binding assays could determine if the coiled-coil domains interact with host proteins or other bacterial components.

How does MPN_010 compare to other DUF16 domain-containing proteins across bacterial species?

Comparative analysis of DUF16 domain-containing proteins reveals evolutionary relationships that may provide functional insights. Phylogenetic analysis has classified the DUF16 family into five subclasses based on N-terminal sequences , suggesting functional diversification during bacterial evolution. Comparing the coiled-coil structures of MPN_010 with those from other bacterial species could reveal conserved structural features that indicate essential functions versus species-specific adaptations that might relate to niche specialization.

Sequence conservation analysis within the DUF16 family shows that certain regions, particularly the 11 highly conserved residues in the shorter coiled-coil and the glutamine-rich region in the longer coiled-coil, are maintained across species . This conservation pattern suggests functional importance for these specific segments. Cross-species functional complementation experiments, where the MPN_010 gene is replaced with DUF16 homologs from other bacteria, could test the functional equivalence of these proteins and reveal species-specific adaptations.

What is the potential of MPN_010 as a target for diagnostic or therapeutic development?

The development of effective vaccines against Mycoplasma pneumoniae has been challenging due to poor immunogenicity and side effects of inactivated or attenuated vaccines . Current research focuses on major antigens like P1 and P30 , but alternative targets such as MPN_010 merit investigation. To evaluate MPN_010's potential as a diagnostic or therapeutic target, researchers should:

• Determine MPN_010 expression levels during infection using quantitative proteomics

• Assess antibody responses to recombinant MPN_010 in MP pneumonia patients

• Evaluate conservation of MPN_010 across MP strains to assess target stability

• Test MPN_010-specific antibodies for effects on bacterial adhesion or viability

• Investigate structural vulnerabilities that could be targeted by small molecules

If MPN_010 plays a role in MP pathogenesis, it could potentially be included in multi-component vaccines or targeted by novel therapeutics. Research on recombinant influenza viruses carrying MP antigens provides a methodological framework that could be adapted for MPN_010 if it proves to be immunogenic or functionally important.

What expression systems are most effective for producing recombinant MPN_010?

Selecting an appropriate expression system for recombinant MPN_010 production requires consideration of protein characteristics and experimental objectives. Based on established protocols for similar bacterial proteins, the following expression systems can be considered:

To optimize expression, researchers should consider:

• Using a tag system (His-tag, GST) for purification while minimizing effects on structure

• Testing different induction conditions (temperature, IPTG concentration)

• Optimizing codon usage for the host organism

• Implementing co-expression of molecular chaperones if folding issues arise

What structural characterization methods are most informative for MPN_010 research?

Building upon the X-ray crystallography data at 1.8Å resolution , additional structural characterization methods can provide complementary insights:

Combining these methods would provide a comprehensive understanding of MPN_010's structural dynamics. For instance, while X-ray crystallography has provided the static structure , NMR could reveal dynamic regions that might be functionally important. SAXS could confirm the homotrimeric state under physiological conditions, while CD spectroscopy could assess structural stability under different pH or temperature conditions.

How can mutagenesis studies help elucidate the function of MPN_010?

Targeted mutagenesis represents a powerful approach to identifying functionally important residues in MPN_010. Based on the structural information available , the following mutagenesis strategies can be employed:

• Alanine scanning of the highly conserved 11 residues in the shorter coiled-coil

• Substitution of key residues in the phenylalanine-rich region to disrupt its unique properties

• Mutations in the glutamine-rich region to test its role in trimerization

• Introduction of helix-breaking residues at specific positions to disrupt coiled-coil formation

• Truncation mutants to isolate the contribution of individual structural elements

Each mutant should be characterized for effects on structure (using CD spectroscopy, size exclusion chromatography), oligomerization (using analytical ultracentrifugation), and function (using binding assays, growth phenotypes). Correlating structural changes with functional effects would establish structure-function relationships for MPN_010.

Additionally, creating chimeric proteins by swapping domains between MPN_010 and related DUF16 proteins from other species could reveal the specificity of functional domains. This approach has proven valuable for understanding the functional evolution of proteins with conserved structural motifs.

What computational tools can predict MPN_010 interactions and functions?

Computational approaches offer valuable insights into potential functions and interactions of MPN_010:

For MPN_010, molecular dynamics simulations could provide insights into the stability of the unique phenylalanine-rich and glutamine-rich regions of the coiled-coil . Analyzing the genomic context of MPN_010 in Mycoplasma pneumoniae could reveal functional associations based on co-regulation or operonic organization. These computational predictions should guide experimental design rather than replace experimental validation.

Machine learning approaches that integrate structural, genomic, and evolutionary data are increasingly powerful for function prediction of uncharacterized proteins like MPN_010. These methods could identify subtle patterns shared with functionally characterized proteins that might not be evident from sequence or structure alone.

How might MPN_010 interact with the human immune system during infection?

Understanding potential interactions between MPN_010 and the human immune system requires consideration of both innate and adaptive immune responses observed during MP infection. Studies of MP pneumonia have demonstrated alterations in cellular immunity, with decreased CD3+ T cells in infected patients . If MPN_010 is exposed to the immune system during infection, it could potentially interact with pattern recognition receptors or serve as an antigen for adaptive immune responses.

Research on MP infections has shown that certain MP proteins induce specific antibody responses. For example, the P1 protein can induce P1-specific IgE, contributing to allergic responses in some individuals . To investigate whether MPN_010 similarly interacts with the immune system, researchers could:

• Test recombinant MPN_010 for activation of immune cells (dendritic cells, macrophages)

• Measure cytokine production in response to MPN_010 exposure

• Assess antibody responses to MPN_010 in patients with confirmed MP infection

• Investigate potential T cell epitopes within the MPN_010 sequence

These studies would clarify whether MPN_010 contributes to the immune dysregulation observed in MP infections, where altered levels of IgG and inflammatory markers have been reported .

Could MPN_010 be incorporated into vaccine development strategies against MP?

Current MP vaccine development focuses on major antigens like P1 and P30, with approaches such as recombinant influenza viruses carrying these antigens showing promise . While MPN_010 is not among the currently targeted antigens, its distinctive structure and potential immunogenicity warrant investigation. To assess MPN_010's potential for vaccine development:

• Determine conservation of MPN_010 across clinical MP isolates to ensure broad protection

• Evaluate immunogenicity of recombinant MPN_010 in animal models

• Test whether anti-MPN_010 antibodies provide protection against MP infection

• Consider MPN_010 as part of a multi-antigen approach if it shows promise

The successful construction of recombinant influenza viruses carrying MP antigens (rFLU-P1a and rFLU-P30a) provides a methodological framework that could potentially be adapted for MPN_010. If MPN_010 proves immunogenic and protective, it could be incorporated into similar vector-based approaches or used in combination with established antigen candidates.

What role might MPN_010 play in MP adhesion to respiratory epithelial cells?

MP pathogenicity depends on adhesion to respiratory epithelial cells, primarily mediated by known adhesins such as the P1 and P30 proteins . While MPN_010 has not been characterized as a primary adhesin, its coiled-coil structure suggests potential involvement in protein-protein interactions that could indirectly contribute to adhesion processes. To investigate this possibility:

• Test whether MPN_010 knockout or knockdown affects MP adhesion to respiratory epithelial cells

• Examine localization of MPN_010 during adhesion using immunofluorescence

• Investigate interactions between MPN_010 and known adhesins using co-immunoprecipitation

• Assess whether anti-MPN_010 antibodies affect MP adhesion capacity

Studies have shown that monoclonal antibodies can substantially reduce or inhibit the adhesion of MP to human respiratory epithelial cells in vitro . If MPN_010 plays a direct or indirect role in adhesion, antibodies targeting it might similarly impact this critical step in MP pathogenesis.

How does MPN_010 expression change during different phases of MP infection?

Understanding the expression dynamics of MPN_010 throughout the infection cycle would provide insights into its potential roles in MP pathogenesis. Researchers could employ the following approaches:

• Quantitative proteomics to measure MPN_010 levels at different infection stages

• Transcriptomic analysis to assess MPN_010 gene expression under various conditions

• Reporter systems (if genetic manipulation is possible) to visualize expression in real-time

• Immunodetection of MPN_010 in clinical samples from different stages of infection

Correlation of MPN_010 expression patterns with specific phases of infection (adhesion, colonization, immune evasion) would provide functional clues. If MPN_010 is upregulated during particular stages, this temporal specificity would suggest stage-specific functions that could be targeted for intervention.