Recombinant Haemophilus influenzae Probable rRNA maturation factor (HI_0004)

What is HI_0004 and why is it significant for research?

HI_0004 is a conserved hypothetical protein from Haemophilus influenzae that has been identified as a probable rRNA maturation factor. Mutagenesis experiments indicate it is an essential gene in H. influenzae, suggesting it plays a crucial role in the organism's survival . The protein belongs to a large family with over 150 homologs spanning from bacteria to humans, indicating strong evolutionary conservation and functional importance . Its structural similarity to eukaryotic matrix metalloproteases (MMPs), particularly in a conserved histidine-rich region, suggests potential hydrolase activity that may be relevant to RNA processing mechanisms .

What is known about the structural characteristics of HI_0004?

The solution structure of HI_0004 has been determined using multidimensional heteronuclear NMR spectroscopy. The protein exhibits an α–β–α sandwich architecture consisting of:

• A central four-stranded β-sheet

• The α2-helix packed against one side of the β-sheet

• Four α-helices (α1, α3, α4, α5) on the other side

• Secondary structure arrangement of β–α–β–α–β–β–α–α–α with a β-strand order of 1243

• Three parallel β-strands (β1, β2, β4) and one antiparallel strand (β3)

The most defined regions correspond with secondary structure elements while loop regions (particularly α1–β2, β3–β4, and α4–α5) display greater conformational flexibility based on 15N-transverse relaxation rates and {1H}-15N steady-state NOE measurements . Additionally, the C-terminal residues 147–154 and N-terminal residues 1–3 are disordered with few detectable inter-residue NOE contacts .

What expression systems and purification methods are recommended for recombinant HI_0004?

For recombinant expression of HI_0004, researchers have successfully used E. coli as the host organism . The following methodology is recommended:

• Clone the hi0004 gene into a suitable expression vector with an affinity tag

• Transform into an E. coli expression strain (e.g., BL21(DE3))

• Culture in appropriate media (LB for unlabeled protein; minimal media with 15N/13C sources for NMR studies)

• Induce expression under optimized conditions

• Lyse cells and purify using affinity chromatography

• Apply size exclusion chromatography for final purification

For NMR studies, the protein should be expressed in minimal media supplemented with labeled compounds such as 15NH4Cl and 13C-glucose . The protein appears to be monomeric in solution based on size exclusion chromatography and NMR line-width characteristics .

What evidence supports HI_0004's role in rRNA maturation?

Several lines of evidence suggest HI_0004's involvement in rRNA maturation:

• Structural similarities to known ribosome biogenesis factors, particularly the presence of conserved histidine residues that could function in RNA processing

• Identification as an essential gene in H. influenzae, consistent with a role in fundamental cellular processes like ribosome biogenesis

• Zinc-binding capacity, which is a common feature among RNA processing enzymes

• Potential relation to factors like Rio2/RIOK2 and SLX9/C21orf70/FAM207A that are known to be involved in ribosome biogenesis and pre-40S subunit export

• Structural homology with metalloproteases suggests potential hydrolytic activity that could function in RNA processing

While direct experimental evidence specifically confirming rRNA maturation activity is still emerging, these characteristics collectively support this functional hypothesis.

How should researchers investigate HI_0004's zinc binding properties and their functional implications?

To comprehensively characterize HI_0004's interaction with zinc, researchers should employ multiple complementary approaches:

• NMR Chemical Shift Perturbation Analysis:

  • Conduct 15N-HSQC experiments with incremental zinc addition

  • Map chemical shift changes onto the 3D structure to identify binding interfaces

  • Previous studies showed significant shift changes in conserved histidines (H114, H118, H124) and adjacent regions including the α4–α5 loop and α5-helix upon zinc addition

• Site-Directed Mutagenesis:

  • Generate single and combination mutants of the conserved histidines

  • Test zinc binding capacity of mutants using spectroscopic methods

  • Assess functional consequences in activity assays

• Biophysical Characterization:

  • Use isothermal titration calorimetry to determine binding affinity and stoichiometry

  • Apply X-ray absorption spectroscopy to characterize the zinc coordination environment

  • Employ circular dichroism to assess structural changes upon zinc binding

• Structural Studies of the Zinc-Bound Form:

  • Determine the solution or crystal structure of zinc-bound HI_0004

  • Focus on conformational changes in the α4–α5 loop and α5-helix regions

  • Compare with the zinc-free structure to identify potential mechanistic insights

Previous work demonstrated that binding of zinc resulted in two distinct sets of peaks in the HSQC spectrum corresponding to free and bound forms in slow exchange on the chemical shift timescale .

What insights can be gained from comparing HI_0004 with its Aquifex aeolicus homolog AQ_1354?

The comparison between HI_0004 and AQ_1354 (36% sequence identity) reveals striking structural differences that provide valuable functional insights:

These differences suggest that HI_0004 may undergo regulated conformational changes upon zinc binding that could serve as a mechanism for activity control . The architectural differences may influence substrate specificity, binding partner interactions, or adaptation to different cellular environments between the two organisms.

What experimental approaches should be prioritized to investigate HI_0004's role in ribosome biogenesis?

To establish HI_0004's role in ribosome biogenesis, researchers should implement these key experimental approaches:

• Ribosome Assembly Analysis:

  • Employ sucrose gradient fractionation to identify which ribosomal subunits or pre-ribosomal particles HI_0004 associates with

  • Use Western blotting to track HI_0004 across gradient fractions

  • Compare sedimentation profiles with known ribosome biogenesis factors like RIOK2

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation with tagged HI_0004 followed by mass spectrometry

  • Use proximity labeling techniques (BioID, APEX) to identify neighboring proteins in vivo

  • Conduct yeast two-hybrid or bacterial two-hybrid screens against ribosome biogenesis factors

• RNA Association Analysis:

  • Implement RNA immunoprecipitation to identify RNA species that interact with HI_0004

  • Use CLIP-seq (Cross-linking immunoprecipitation followed by sequencing) to map binding sites on pre-rRNAs

  • Perform in vitro binding assays with synthetic pre-rRNA fragments

• Functional Depletion Studies:

  • Develop conditional expression systems for HI_0004 in H. influenzae

  • Analyze pre-rRNA processing intermediates by Northern blotting after depletion

  • Examine polysome profiles to assess impact on ribosome assembly and translation

• Structural Biology Approaches:

  • Determine cryo-EM structures of HI_0004 in complex with pre-ribosomal particles

  • Map HI_0004's binding site in the context of ribosome assembly

Researchers should consider parallels with established ribosome biogenesis factors like Rio2/RIOK2, which associates with pre-40S particles and functions in ribosome export pathways .

How should in vitro assays be designed to test HI_0004's potential hydrolase activity?

Designing robust in vitro assays for HI_0004's putative hydrolase activity requires careful consideration of multiple parameters:

• Substrate Selection:

  • Pre-rRNA segments from regions requiring processing

  • Synthetic RNA oligonucleotides with fluorescent reporters

  • Generic metalloprotease substrates as positive controls

  • Consider both sequence-specific and structure-specific substrates

• Reaction Conditions Optimization:

  • Metal cofactors: Test various concentrations of Zn2+ (0.1-10 mM) and other divalent metals (Mg2+, Mn2+)

  • pH range: Examine pH 6.0-8.5 in appropriate buffer systems

  • Ionic strength: Vary NaCl concentration (50-300 mM)

  • Temperature: Test physiologically relevant range (25-37°C)

• Essential Controls:

  • Metal chelators (EDTA, 1,10-phenanthroline) to confirm metal dependence

  • Heat-inactivated enzyme

  • HI_0004 histidine mutants (H114A, H118A, H124A)

  • Known RNA processing enzymes as positive controls

• Detection Methods:

  • Gel electrophoresis with radioisotope or fluorescent labeling

  • FRET-based continuous assays for real-time monitoring

  • Mass spectrometry to identify precise cleavage sites

  • Primer extension to map 5′ ends generated by processing

• Kinetic Analysis:

  • Determine Michaelis-Menten parameters (Km, kcat, kcat/Km)

  • Assess substrate specificity using comparative kinetics

  • Investigate potential cooperativity or allostery in zinc binding

Previous studies have shown that zinc binding induces conformational changes in HI_0004, particularly in regions containing the conserved histidines, which should be considered when interpreting activity results .

How can researchers address inconsistencies between structural predictions and experimental observations for HI_0004?

To resolve discrepancies between predicted and experimental structures of HI_0004, researchers should implement a systematic investigative strategy:

• Integrative Structural Biology Approaches:

  • Combine data from multiple techniques (NMR, X-ray, SAXS, cryo-EM)

  • Use ensemble methods to represent conformational heterogeneity

  • Apply integrative modeling software (IMP, HADDOCK) to merge diverse data types

  • Validate models against independent experimental measurements

• Dynamics Characterization:

  • Perform NMR relaxation dispersion experiments to identify regions undergoing conformational exchange

  • Employ hydrogen/deuterium exchange mass spectrometry to map structural stability

  • Use molecular dynamics simulations to sample the conformational landscape

  • Apply normal mode analysis to identify potential large-scale motions

• Metal-Binding Site Validation:

  • Use paramagnetic NMR with cobalt substitution for zinc

  • Apply site-directed spin labeling to measure distances between key residues

  • Perform mutagenesis of putative coordinating residues with functional testing

  • Compare with structurally similar metalloproteins

• Experimental Conditions Analysis:

  • Systematically vary buffer conditions, pH, salt concentration to identify factors affecting conformation

  • Test different protein constructs (truncations, extensions) to assess domain influences

  • Examine potential oligomerization or aggregation effects

• Computational Approaches:

  • Apply model validation tools (MolProbity, PROCHECK)

  • Use conformational sampling methods to explore alternative structures

  • Implement enhanced sampling techniques to identify low-energy conformations

  • Analyze sequence conservation patterns in the context of structural models

The observed differences between solution NMR structure of HI_0004 and crystal structure of AQ_1354 suggest conformational plasticity that may be functionally relevant .

What methodologies are recommended for analyzing NMR relaxation data of HI_0004?

Comprehensive analysis of NMR relaxation data for HI_0004 requires sophisticated methodological approaches:

Previous studies have identified specific mobile regions in HI_0004, including loops α1–β2, β3–β4, and α4–α5, based on 15N-transverse relaxation rates and {1H}-15N steady-state NOE measurements . These regions may be particularly important for conformational changes associated with zinc binding and potential substrate interactions.

How should researchers design experiments to establish HI_0004's specific rRNA processing activity?

To definitively establish HI_0004's role in rRNA processing, researchers should implement a comprehensive experimental strategy:

• In Vitro RNA Processing Assays:

  • Generate synthetic pre-rRNA substrates corresponding to known processing sites

  • Incubate purified recombinant HI_0004 with these substrates under optimized conditions

  • Include variants with and without zinc to assess metal dependence

  • Analyze processing products using gel electrophoresis, primer extension, and sequencing

  • Include appropriate positive controls (known processing enzymes) and negative controls

• Substrate Specificity Analysis:

  • Test various pre-rRNA segments to identify preferred substrates

  • Compare activity on RNA substrates with different secondary structures

  • Perform competition assays to determine substrate preference hierarchy

  • Map exact cleavage sites using primer extension or RNA sequencing

• Cellular RNA Processing Analysis:

  • Develop conditional depletion systems for HI_0004 in H. influenzae

  • Analyze pre-rRNA processing intermediates by Northern blotting after depletion

  • Perform pulse-chase labeling experiments to track rRNA maturation kinetics

  • Use RNA-seq to comprehensively identify affected RNA species

• Structure-Function Studies:

  • Generate structure-guided mutants affecting the potential active site

  • Focus particularly on the conserved histidines (H114, H118, H124)

  • Test activity of mutants in both in vitro and in vivo assays

  • Correlate structural features with processing activity

• Interaction Studies with Ribosome Assembly Machinery:

  • Examine HI_0004's association with pre-ribosomal particles

  • Test for interactions with other known ribosome biogenesis factors

  • Perform reconstitution experiments with defined components

  • Use cryo-EM to visualize HI_0004 in the context of pre-ribosomal particles

The experimental design should consider HI_0004's potential relationship to other ribosome biogenesis factors like RIOK2, which has established roles in pre-40S ribosomal subunit maturation .