Recombinant Arabidopsis thaliana Probable inactive receptor kinase At2g26730 (At2g26730)

What is At2G26730 and how is it classified within plant receptor kinases?

At2G26730 is classified as a probable inactive receptor kinase in Arabidopsis thaliana. It belongs to the broader family of receptor kinases but is categorized as "probable inactive" due to structural features suggesting limited or absent catalytic activity. This classification places it within the growing category of pseudokinases - proteins with kinase-like domains that lack one or more conserved residues necessary for catalytic function. Pseudokinases like At2G26730 represent approximately 10-15% of the kinome in various organisms and are increasingly recognized for their non-catalytic regulatory roles in signaling pathways .

Methodologically, classification of At2G26730 as a pseudokinase involves sequence analysis to identify deviations in conserved catalytic motifs, structural predictions, and experimental verification of kinase activity using recombinant proteins. Researchers typically assess ATP binding and phosphotransfer capability using radioactive ATP incorporation assays or phospho-specific antibodies to determine if the protein can function as an active kinase.

What are the structural features of At2G26730?

At2G26730 is a full-length protein spanning amino acids 24-658, containing receptor-like domains in its structure . Like other receptor kinases, it likely possesses an extracellular domain involved in ligand perception, a transmembrane domain, and a cytoplasmic kinase-like domain. The protein can be recombinantly expressed with a His-tag to facilitate purification and functional studies .

The predicted structural features of At2G26730 can be analyzed through computational approaches like homology modeling based on crystallized receptor kinase structures. For experimental structural characterization, researchers employ techniques such as X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy, though these require significant quantities of purified protein and can be challenging due to the membrane-associated nature of receptor kinases.

How does At2G26730 compare with other characterized pseudokinases in plants?

At2G26730 shares functional similarities with other characterized pseudokinases in Arabidopsis, such as the subgroup VIII receptor-like cytoplasmic kinases (RLCKs) MAZ and CARK7, which also demonstrate limited or absent catalytic activity in vitro . Similar to these proteins, At2G26730 likely functions through protein-protein interactions rather than phosphorylation-dependent signaling.

Comparative analysis of At2G26730 with other pseudokinases typically involves sequence alignment of kinase domains, phylogenetic analysis, and comparison of conservation patterns in catalytic motifs. Functional comparison requires parallel experimental approaches such as protein-protein interaction studies, subcellular localization, and phenotypic analysis of corresponding mutants.

What expression systems are optimal for recombinant At2G26730 production?

Recombinant At2G26730 has been successfully expressed in E. coli as a His-tagged full-length protein (amino acids 24-658) . For research applications requiring properly folded and post-translationally modified protein, several expression systems can be considered:

The choice between these systems should be guided by the specific experimental needs, balancing protein yield with biological relevance and post-translational modification requirements.

What purification strategies yield functional At2G26730 for biochemical studies?

Purification of recombinant At2G26730 typically leverages affinity chromatography based on the His-tag . A comprehensive purification protocol would include:

• Affinity chromatography: Using Ni-NTA or cobalt-based resins for His-tagged proteins

• Size exclusion chromatography: To separate monomeric protein from aggregates

• Ion exchange chromatography: For final polishing and removal of contaminants

For membrane-associated proteins like receptor kinases, inclusion of appropriate detergents or amphipols during purification is crucial to maintain protein solubility and native conformation. Typical detergents include n-dodecyl-β-D-maltoside (DDM) or digitonin at concentrations above their critical micelle concentration.

The protein's functional state should be verified through biophysical methods such as circular dichroism to assess secondary structure, and dynamic light scattering to confirm monodispersity.

What experimental approaches can determine if At2G26730 truly lacks kinase activity?

Despite classification as a "probable inactive receptor kinase," experimental validation of At2G26730's catalytic status is essential. Methodological approaches include:

• In vitro kinase assays: Purified recombinant At2G26730 can be incubated with radioactive ATP (γ-32P-ATP) and potential substrates to detect phosphotransfer activity. Parallel assays with known active kinases serve as positive controls.

• ATP binding assays: Techniques such as thermal shift assays or fluorescent ATP analogs can assess whether At2G26730 binds ATP, a prerequisite for kinase activity.

• Structural analysis of the ATP-binding pocket: Computational modeling and mutagenesis of key residues in the catalytic site can identify deviations from consensus motifs required for kinase activity.

Similar approaches have been applied to other pseudokinases such as MAZ and CARK7, confirming their lack of catalytic activity despite structural similarity to active kinases .

How might At2G26730 function if it lacks catalytic activity?

Research on other plant pseudokinases suggests several potential non-catalytic functions for At2G26730:

• Scaffolding function: The protein may serve as a molecular scaffold, facilitating the assembly of signaling complexes, similar to how group 1 REMs function as scaffold proteins in viral immunity .

• Allosteric regulation: At2G26730 might modulate the activity of interacting proteins through conformational changes rather than phosphorylation.

• Competition for substrates: It may compete with active kinases for binding to substrates, thereby regulating their phosphorylation status.

• Decoy receptors: As suggested for other inactive receptor kinases, At2G26730 might function as a decoy receptor that sequesters ligands without triggering downstream signaling.

To investigate these possibilities, co-immunoprecipitation coupled with mass spectrometry can identify At2G26730 interacting partners, while bimolecular fluorescence complementation or FRET-based approaches can validate these interactions in planta. Studies with MAZ have demonstrated that variants incapable of kinase activity can still complement corresponding mutants, suggesting important non-catalytic roles in plants .

What role might At2G26730 play in plant immune signaling?

Based on studies of similar receptor kinases in Arabidopsis, At2G26730 may function in plant immune signaling pathways. Several lines of evidence support this hypothesis:

• Many receptor kinases and pseudokinases in Arabidopsis, including the RLCKs MAZ and CARK6, function as regulators of immune responses .

• Plant pattern recognition receptors (PRRs) such as FLS2 and EFR (leucine-rich repeat receptor kinases) and RLP23 and RLP42 (leucine-rich repeat receptor proteins) detect microbial patterns and activate innate immunity .

• Pseudokinases often function as regulatory components in immune signaling cascades, as exemplified by BIK1, which functions as a positive regulator of receptor kinase signaling but a negative regulator of receptor protein-mediated immunity .

To investigate At2G26730's role in immunity, researchers should consider pathogen infection assays using loss-of-function mutants, measuring typical immune responses such as reactive oxygen species production, callose deposition, and expression of defense-related genes. Complementation with wild-type and catalytically inactive variants can distinguish between catalytic and non-catalytic functions.

How might At2G26730 interact with calcium-dependent signaling pathways?

Calcium signaling plays a crucial role in plant immune responses, and At2G26730 might interact with calcium-dependent protein kinases (CPKs) similar to other regulatory kinases. For example:

• Subgroup VIII RLCKs like MAZ and CARK7 associate with the calcium-dependent protein kinase CPK28 in planta .

• CPK28 phosphorylates both MAZ and CARK7 on multiple residues in regions critical for protein kinase activation .

• Group 1 REMs have been identified as in vitro substrates of CPK3, establishing a connection between calcium signaling and membrane-associated regulatory proteins in immune responses .

To investigate At2G26730's potential involvement in calcium-dependent signaling, co-immunoprecipitation with CPKs followed by mass spectrometry analysis can identify potential phosphorylation sites. Calcium imaging in cells expressing At2G26730 during immune elicitation could reveal functional connections to calcium signaling dynamics.

What methodologies are most effective for identifying At2G26730 interacting partners?

To comprehensively characterize the interactome of At2G26730, several complementary approaches should be employed:

• Immunoprecipitation coupled to mass spectrometry (IP-MS): This approach has successfully identified interactors of related proteins, such as CPK3 being identified as an interactor of REM1.2 in Arabidopsis . For At2G26730, expressing tagged versions (His, FLAG, or GFP) in Arabidopsis followed by IP-MS under both basal and stimulated conditions can reveal context-dependent interactions.

• Yeast two-hybrid (Y2H) screening: While traditional Y2H may be challenging for membrane proteins, split-ubiquitin Y2H systems are adapted for membrane-associated proteins like receptor kinases.

• Bimolecular fluorescence complementation (BiFC): This technique allows visualization of protein-protein interactions in planta by fusing complementary fragments of a fluorescent protein to potential interacting partners.

• Förster resonance energy transfer (FRET): This approach can detect protein interactions with high spatial resolution in living cells, particularly useful for membrane-localized proteins.

• Protein microarrays: Arrays containing purified Arabidopsis proteins can be probed with recombinant At2G26730 to identify direct binding partners.

To minimize false positives, interactions should be validated using multiple independent techniques and controls for non-specific binding.

How might At2G26730 participate in homo- and hetero-dimerization?

Receptor kinases commonly function through ligand-induced dimerization, and even catalytically inactive kinases can participate in these interactions. Based on related proteins:

• Subgroup VIII RLCKs demonstrate homo- and hetero-dimerization capabilities, with evidence supporting interactions between CARK7 and MAZ .

• The chitin receptor AtCERK1 forms homodimers upon binding to long-chain chitin oligomers, leading to activation of its intracellular kinase domain .

• Some receptor complexes involve both active and inactive kinase partners, where the inactive partner serves a regulatory function.

To investigate At2G26730 dimerization, approaches such as chemical crosslinking followed by SDS-PAGE, size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), and in-cell FRET between differently tagged variants can be employed. Mapping the dimerization interface through mutagenesis can further elucidate the structural basis of these interactions.

How can CRISPR-Cas9 technology be optimized for studying At2G26730 function?

CRISPR-Cas9 technology offers powerful approaches for studying At2G26730 function through various strategies:

• Gene knockout: Complete deletion or disruption of At2G26730 to assess loss-of-function phenotypes. This approach has been successfully used to generate quadruple mutants combining mutations in REM1.2, REM1.3, REM1.4, and CPK3 .

• Domain-specific mutations: Targeted mutations in specific functional domains (e.g., the kinase domain or potential dimerization interfaces) can distinguish domain-specific functions.

• Base editing: Precise modification of specific amino acids to alter potential phosphorylation sites or catalytic residues without disrupting protein expression.

• Knock-in tags: Insertion of fluorescent or affinity tags at the genomic locus ensures expression at physiological levels.

When designing CRISPR strategies for At2G26730, researchers should consider potential genetic redundancy with related proteins, as observed with REM1.2 and REM1.3, which share 95% of their interactome and appear functionally redundant .

What high-throughput approaches can advance understanding of At2G26730 function?

Several high-throughput methodologies can accelerate functional characterization of At2G26730:

• Phosphoproteomics: Comparing phosphorylation profiles between wild-type and At2G26730 mutant plants under various conditions can identify downstream signaling components, even if At2G26730 itself lacks kinase activity.

• Transcriptomics: RNA-seq analysis comparing gene expression patterns between wild-type and mutant plants can reveal pathways influenced by At2G26730, similar to how transcriptomic analysis revealed that RLP23-regulated genes represent only a fraction of those differentially expressed upon FLS2 activation .

• Interactome mapping: High-throughput protein-protein interaction screens using techniques like proximity labeling (BioID or TurboID) can identify proteins that interact with At2G26730 in their native cellular environment.

• Chemical genetics: Screening chemical libraries for compounds that affect At2G26730 function or its interaction with partners can provide both research tools and potential agricultural applications.

These approaches should be combined with targeted validation experiments to confirm high-throughput findings and establish mechanistic understanding.