Recombinant Arabidopsis thaliana B3 domain-containing protein At2g31862 (At2g31862)

What is the B3 domain-containing protein At2g31862?

The B3 domain-containing protein At2g31862 is a transcription factor from Arabidopsis thaliana that belongs to the plant-specific B3 superfamily. This protein is characterized by its B3 DNA-binding domain, which is involved in the recognition of specific DNA sequences for transcriptional regulation. According to the available data, At2g31862 is a full-length protein comprising 174 amino acids and is localized to the nucleus, consistent with its predicted function in transcriptional regulation . The protein has been recombinantly produced with the product code CSB-MP718792DOA and is cataloged in UniProt under the accession number Q6DSS2 .

What is the amino acid sequence of At2g31862?

The full amino acid sequence of the At2g31862 protein consists of 174 residues as follows:

MWVNLSCMCHIVDKLLELNLRRWNMRSTSIYVLASRWKKVVSDNTLIEGQRIRLWSFHSLAKLYIALVPLDPAPAPTLAILLAPAPTPSSPPVVTRDSDELYISHADAQEEGDRILPVHADNDWECLNLLAKVSEETTCLEVSQEANRSSLVSDTELDLELRLSLPGKNSYVM

This sequence contains the characteristic B3 domain motif that defines this family of plant transcription factors. Understanding this sequence is essential for structural analyses, functional studies, and the design of experiments involving this protein.

How do B3 domains recognize and bind to DNA?

B3 domains interact with DNA through specific structural elements that facilitate recognition and binding. Research on related B3 domain proteins has revealed that key residues at positions 64, 66, and 69 play critical roles in DNA binding specificity . These positions show variation among different B3 domain proteins, contributing to their diverse functions:

Position 64 can contain various amino acids (R, Q, or K) that influence DNA binding through different hydrogen bonding patterns. For example, in proteins like VAL1 and ABI3, this residue forms a hydrogen bond with the backbone carbonyl of amino acid D46, while in proteins like LEC2 and FUS3, K64 extends toward the phosphate backbone .

Position 66 typically contains positively charged residues (R or K) that interact electrostatically with the DNA backbone. The side chain length affects the proximity of the protein backbone to the DNA, with R66 (as in VAL1) limiting proximity compared to K66 (as in LEC2) .

Position 69 can contain either serine or proline, with serine enabling hydrogen bonding with the DNA phosphate backbone while proline lacks this capacity. This difference significantly impacts binding affinity, as seen in the pronounced effect of S69P substitutions on B3 domain activity .

What structural features distinguish At2g31862 from other B3 domain proteins?

While specific structural data for At2g31862 is limited in the provided sources, comparative analysis with other B3 domain proteins reveals potential distinctive features. B3 domains generally adopt a seven-stranded β-sheet structure arranged in an open β-barrel configuration with two α-helices . The structural distinction between different B3 domain proteins primarily lies in the specific amino acid composition at key positions that affect DNA binding.

In LEC2, for instance, the distance between the peptide backbone and nearest DNA phosphate is approximately 3.2 Å, whereas in VAL1 this distance is 5.75 Å and in FUS3 it is 4.67 Å . These differences in backbone proximity are accommodated by variations in hydrogen bonding patterns and side chain conformations, particularly at positions 64, 66, and 69. Determining the specific residues at these positions in At2g31862 would provide insight into its unique structural properties and DNA binding preferences.

Experimental approaches such as X-ray crystallography or NMR spectroscopy would be necessary to definitively characterize the three-dimensional structure of At2g31862 and its mode of interaction with DNA.

How does At2g31862 compare to other characterized B3 domain proteins?

Comparative analysis of At2g31862 with other B3 domain proteins reveals both similarities and potential functional divergence. The following table summarizes key features of At2g31862 and selected well-characterized B3 domain proteins:

This comparison highlights the structural diversity within the B3 domain family and suggests that At2g31862 may have evolved specific DNA binding preferences that contribute to its unique biological function in Arabidopsis.

What are the optimal conditions for reconstituting and storing recombinant At2g31862?

For optimal reconstitution of lyophilized recombinant At2g31862 protein, researchers should follow these methodological guidelines:

• Centrifuge the vial briefly before opening to bring contents to the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) before aliquoting and storing at -20°C/-80°C .

The storage conditions significantly impact protein stability and shelf life:

These recommendations are based on general principles for maintaining protein stability and activity, with the understanding that repeated freeze-thaw cycles can compromise protein integrity and function .

What methods can be used to study the DNA binding specificity of At2g31862?

Multiple complementary approaches can be employed to characterize the DNA binding specificity of At2g31862:

Electrophoretic Mobility Shift Assay (EMSA) is a fundamental technique for detecting protein-DNA interactions. In this approach, purified At2g31862 is incubated with labeled DNA fragments containing potential binding sites, and the resulting complexes are resolved by electrophoresis. A shift in DNA mobility indicates binding, allowing researchers to identify sequence preferences and binding affinity.

Chromatin Immunoprecipitation (ChIP) can be used to identify genomic binding sites of At2g31862 in vivo. This technique involves crosslinking proteins to DNA in living cells, fragmenting the chromatin, and immunoprecipitating At2g31862 along with its bound DNA fragments. The isolated DNA can then be analyzed by sequencing (ChIP-seq) to generate a genome-wide map of binding sites.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) allows determination of the consensus DNA binding sequence preferred by At2g31862. This iterative selection process enriches for high-affinity binding sequences from a random oligonucleotide pool.

Structural approaches such as X-ray crystallography or NMR spectroscopy provide atomic-level details of protein-DNA interactions. These methods could reveal how specific residues in At2g31862 contact DNA and how these interactions contribute to binding specificity, particularly in comparison to other B3 domain proteins like those characterized in the provided sources .

How can researchers verify the functionality of purified At2g31862 protein?

To ensure that purified At2g31862 retains its biological activity, researchers should implement a comprehensive validation strategy:

First, structural integrity should be assessed using biophysical techniques such as circular dichroism spectroscopy to confirm proper secondary structure formation, and size exclusion chromatography to verify the monomeric state or appropriate oligomerization status.

DNA binding activity can be verified using several complementary methods:

• Electrophoretic Mobility Shift Assay (EMSA) with predicted target sequences

• Fluorescence anisotropy to measure binding affinity and kinetics

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) for quantitative analysis of binding interactions

Specificity controls are essential to confirm authentic biological activity:

• Competition assays with specific and non-specific DNA sequences

• Mutational analysis of key residues predicted to affect DNA binding (especially positions 64, 66, and 69 based on studies of related B3 domains)

• Comparison with known active B3 domain proteins as positive controls

Each validation method should include appropriate controls to ensure reliable results and confirm that the purified protein maintains its native functional properties.

How might At2g31862 be involved in plant stress response pathways?

While direct evidence of At2g31862's role in stress responses is limited in the provided sources, its potential involvement can be hypothesized based on contextual information. At2g31862 appears as a candidate gene potentially related to suppressor mutants of stop1 in Arabidopsis . STOP1 is a master transcription factor controlling expression of aluminum tolerance genes and processes involved in low pH resistance, and loss-of-function stop1 mutants are extremely sensitive to low pH and aluminum stresses .

The identification of At2g31862 in this context suggests several potential roles in stress response pathways:

• It may function as a transcriptional regulator of genes involved in acid soil stress adaptation.

• It could participate in aluminum detoxification or exclusion mechanisms.

• It might be involved in general abiotic stress signaling pathways that intersect with proton and aluminum stress responses.

To investigate these hypotheses, researchers could:

• Analyze expression patterns of At2g31862 under various stress conditions, particularly low pH and aluminum toxicity

• Generate and characterize knockout or overexpression lines

• Identify target genes through ChIP-seq or RNA-seq approaches

• Assess phenotypic responses of mutant plants to relevant stresses

• Investigate protein-protein interactions with known components of stress signaling pathways, including STOP1

What molecular mechanisms might underlie the potential role of At2g31862 in STOP1-mediated stress responses?

Based on the identified suppressor mutants of stop1 (tps mutants) described in the research, several molecular mechanisms could explain how At2g31862 might function in STOP1-mediated stress responses:

STOP1 regulates the expression of key aluminum tolerance genes such as ALMT1 (encoding a malate transporter) and MATE (encoding a citrate transporter), which mediate organic acid exudation from roots to chelate toxic Al³⁺ in the rhizosphere . The study found that some tps mutations partially rescue the hypersensitive phenotypes of stop1 to both low pH and aluminum stress, while others enhance resistance only to low pH stress .

The research demonstrated that certain tps mutations increased Al-induced ALMT1 expression and associated malate exudation in the stop1 background, suggesting activation of STOP1-independent pathways . As a B3 domain transcription factor, At2g31862 could potentially:

• Act as a repressor of ALMT1 or other stress response genes that is normally regulated by STOP1

• Function in an alternative transcriptional pathway that becomes activated when STOP1 is absent

• Interact with other transcription factors involved in stress responses, modifying their activity

• Compete with STOP1 for binding to promoter regions of target genes

Experimental approaches to investigate these mechanisms would include:

• Chromatin immunoprecipitation to identify direct target genes

• Yeast one-hybrid or electrophoretic mobility shift assays to characterize DNA binding sites

• Protein-protein interaction studies to identify potential partners

• Transcriptome analysis of At2g31862 knockout or overexpression lines under stress conditions

How can site-directed mutagenesis be used to investigate the DNA binding specificity of At2g31862?

Site-directed mutagenesis offers a powerful approach to systematically investigate how specific amino acid residues contribute to the DNA binding specificity of At2g31862. Based on structural studies of related B3 domains, a targeted mutagenesis strategy would focus on key residues known to influence DNA binding:

Positions 64, 66, and 69 are particularly important for determining DNA binding specificity in B3 domains . For instance, in related B3 domains:

• Position 64 can contain K, Q, or R, affecting hydrogen bonding patterns with DNA

• Position 66 typically contains R or K, influencing the proximity of the protein backbone to DNA

• Position 69 can be S or P, with S enabling hydrogen bonding with the DNA phosphate backbone while P lacks this capacity

A comprehensive mutagenesis strategy would include:

• Single mutations at key positions to convert residues to those found in other B3 domain proteins (e.g., if At2g31862 has K64, mutate to Q64 or R64)

• Combined mutations to recreate binding patterns of specific B3 domain proteins (e.g., introduce the exact combination found in LEC2 or VAL1)

• Alanine scanning mutagenesis of the B3 domain to identify additional residues critical for DNA binding

• Chimeric constructs that swap regions between At2g31862 and other B3 domain proteins

Each mutant protein would be purified and characterized for DNA binding using:

• EMSAs to assess binding affinity

• DNase I footprinting to determine precise contact sites

• Binding site selection methods to identify altered sequence preferences

• Structural analyses to visualize changes in protein-DNA interactions

This approach would provide insights into the molecular determinants of At2g31862's binding specificity and how it compares to other members of the B3 domain family.

What key amino acid residues determine the DNA binding specificity of B3 domains?

Research on related B3 domain proteins has identified specific amino acid positions that play critical roles in determining DNA binding specificity. The following table summarizes these key positions and their effects on DNA binding:

These residue variations create distinct patterns of interaction with DNA that contribute to binding specificity. For example, in the LEC2 B3 domain, the distance between the peptide backbone and the nearest DNA phosphate is approximately 3.2 Å, compared to 5.75 Å in VAL1 and 4.67 Å in FUS3 . This closer proximity in LEC2 is accommodated by the formation of a hydrogen bond between the amino nitrogen of S69 and a phosphate of the DNA backbone .

Identifying the specific residues at these positions in At2g31862 would provide valuable insights into its DNA binding preferences and functional specificity.

How do B3 domains achieve both affinity and specificity in DNA recognition?

B3 domains employ multiple molecular mechanisms to achieve both high affinity and specificity in DNA recognition. Research on related B3 domain proteins reveals that these mechanisms involve a combination of direct base recognition and indirect readout of DNA structural properties:

Hydrogen bonding networks further stabilize protein-DNA complexes. For instance, when S69 is present (as in LEC2 and FUS3), its amino nitrogen forms a hydrogen bond with a phosphate of the DNA backbone . In contrast, P69 (found in VAL1 and ABI3) lacks this hydrogen bonding capacity, resulting in different binding characteristics.

Residue 64 also plays an important role in DNA interaction, although the precise mechanism varies between B3 domains. In ABI3 and VAL1, R64 and Q64 respectively form a hydrogen bond with the backbone carbonyl of amino acid D46, while in LEC2 and FUS3, K64 extends toward the phosphate backbone .

These various interaction patterns create distinct binding surfaces that recognize specific DNA sequences with differing affinities. The collective action of these residues, along with other structural elements of the B3 domain, enables precise discrimination between target and non-target DNA sequences.

What structural accommodations occur at the protein-DNA interface during B3 domain binding?

When B3 domains bind to DNA, several structural accommodations occur at the protein-DNA interface to optimize interactions. Analysis of related B3 domain structures reveals these key adaptations:

Side chain conformations can also adjust to optimize interactions. In FUS3, an intermediate backbone separation of 8.56 Å is associated with a retracted conformation of the R66 side-chain relative to VAL1 R66 . This conformational flexibility allows fine-tuning of electrostatic interactions with the DNA backbone.

Hydrogen bonding patterns vary significantly between different B3 domains. For example, in ABI3 B3 domain models, the R64 side-chain consistently forms a hydrogen bond with the backbone carbonyl of amino acid D46. The amide nitrogen of Q64 in VAL1 forms an analogous hydrogen bond, while the K64 side-chains of LEC2 and FUS3 extend toward the phosphate backbone instead .

These structural accommodations highlight the adaptability of B3 domains in optimizing interactions with DNA targets, contributing to their diverse functional roles in plant transcriptional regulation.

What are the most promising future research directions for At2g31862?

Future research on At2g31862 should focus on several key areas to fully elucidate its biological function and molecular mechanisms. First, comprehensive characterization of its DNA binding specificity using techniques such as ChIP-seq, SELEX, and structural studies would provide fundamental insights into its targets and mode of action. Genetic approaches, including the generation and phenotypic analysis of knockout and overexpression lines, would help establish its physiological roles, particularly in stress response pathways suggested by its identification in the context of stop1 suppressor mutants .

Investigation of potential protein-protein interactions would reveal how At2g31862 is integrated into broader transcriptional regulatory networks. This could include identifying potential coactivators, corepressors, or other transcription factors that cooperate with At2g31862 to regulate gene expression. Additionally, comparative studies with other B3 domain proteins would enhance understanding of functional diversification within this important plant-specific transcription factor family.

Given its potential involvement in acid soil stress responses, investigating At2g31862's role in agricultural contexts could lead to practical applications. Engineering plants with modified At2g31862 expression or activity might enhance crop resistance to acid soils and aluminum toxicity, addressing significant agricultural challenges worldwide.

How might understanding At2g31862 function contribute to plant stress tolerance research?

Understanding At2g31862 function could significantly advance plant stress tolerance research in several ways. Its identification as a candidate gene in the context of stop1 suppressor mutants suggests it may be involved in acid soil stress responses, which are major limiting factors for crop production worldwide . If At2g31862 regulates genes involved in proton or aluminum tolerance, manipulating its expression or activity could potentially enhance crop performance on acidic soils.

The study of At2g31862 could also provide broader insights into transcriptional regulatory networks governing plant responses to environmental stresses. As a B3 domain transcription factor, it likely controls multiple target genes, potentially revealing novel stress response pathways that could be exploited for crop improvement.

Furthermore, comparative analysis of At2g31862 function across different plant species might identify conserved and divergent aspects of stress response mechanisms, contributing to our fundamental understanding of plant adaptation to adverse environmental conditions. This knowledge could inform breeding programs and biotechnological approaches aimed at developing crops with enhanced stress resilience, addressing the growing challenges of climate change and food security.