Recombinant Calcium-dependent protein kinase 1 (CPK1), partial

What is the structure and function of CDPK1?

CDPK1 is a calcium-regulated enzyme with an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain containing EF-hands that bind calcium ions. The binding of calcium to the EF-hands modulates protein conformation, inducing kinase activity. In Plasmodium falciparum, CDPK1 has a threonine (T145) at the gatekeeper position in the wild-type enzyme. The protein undergoes significant conformational changes upon calcium binding, transitioning from an inactive to an active state .

In apicomplexan parasites like Toxoplasma gondii and Plasmodium species, CDPKs function as major transducers of calcium signaling. CDPK1 specifically plays an essential role in the invasion of host cells by these parasites, making it a potential target for antimalarial and antiparasitic drug development .

How does calcium regulate CDPK1 activity?

Calcium ions bind to the EF-hands in the calmodulin-like domain of CDPK1, triggering conformational changes that activate the kinase. Fluorescence emission spectroscopy shows that CDPK1 undergoes quenching with a 5 nm red shift upon calcium addition, indicating significant conformational alterations in its tertiary structure .

Studies have revealed a biphasic calcium binding curve with two distinct binding constants (1.29 μM and 120 μM in one study; 0.027 μM and 1.7 μM in isothermal calorimetric titration), suggesting heterogeneity in the calcium-binding properties of the EF-hands . This calcium binding increases the α-helical content of CDPK1 (from 75% to 81% in CaCDPK1) and causes exposure of hydrophobic surfaces as demonstrated by increased fluorescence of ANS (8-anilinonaphthalene-1-sulfonic acid) .

What expression systems are typically used for recombinant CDPK1 production?

For recombinant CDPK1 production, researchers commonly use bacterial expression systems with E. coli. Based on the search results, the recombinant proteins are often expressed as GST-fusion proteins to facilitate purification. For instance, in studies of PfCDPK1, researchers expressed wild-type and mutant versions as GST-fusion proteins with molecular weights around 87 kDa, which were detected with both anti-GST and anti-PfCDPK1 sera to confirm their identity .

When designing expression constructs, it's important to consider whether the full-length protein or specific domains are needed for the study. Some research focuses on the interaction between the kinase domain and the calcium-activated domain, which may require expression of separate domains or introduction of specific cleavage sites .

How do mutations in the gatekeeper residue affect CDPK1 function and inhibitor sensitivity?

Mutations in the gatekeeper residue of CDPK1 can significantly impact both enzyme function and inhibitor sensitivity. The replacement of the wild-type threonine gatekeeper residue (T145 in PfCDPK1) with methionine (T145M) reduces transphosphorylation activity by approximately 47% . Despite this reduction in activity, parasites with this mutation can compensate through alternative mechanisms.

Other gatekeeper mutations (glycine, alanine, and tyrosine) have shown highly diminished kinase activity compared to wild-type, though they still retain calcium-dependent activation .

What compensatory mechanisms emerge in response to CDPK1 inhibition or mutation?

Parasites demonstrate remarkable adaptability to CDPK1 inhibition or mutation through several compensatory mechanisms:

• Upregulation of related kinases: In CDPK1 T145M mutant parasites, transcript levels of CDPK5 and CDPK6 are significantly upregulated, suggesting these kinases may compensate for decreased CDPK1 activity, particularly during host cell invasion .

• PKG-mediated compensation: CDPK1 T145M parasites show increased sensitivity to compound 2 (C2), a specific inhibitor of protein kinase G (PKG). This suggests that PKG activity may compensate for reduced CDPK1 function, either directly or by regulating other kinases .

• Structural adaptations: In studies of Toxoplasma gondii CDPK1, mutations like H201Q (replacing histidine with glutamine in the activation loop) and L96P (replacing leucine with proline on helix αC) have been observed. These mutations affect the stabilization of the inactive conformation, potentially influencing kinase activation dynamics .

These compensatory mechanisms highlight why targeting a single kinase may be insufficient for effective antimalarial therapy and suggests that dual-targeting approaches might be more effective in preventing resistance development .

What is the role of autophosphorylation in CDPK1 regulation?

Autophosphorylation is a critical regulatory mechanism for CDPK1 activity. Mass spectrometry analysis has revealed multiple autophosphorylation sites on PfCDPK1, some of which are strategically located and play crucial roles in kinase activation .

These autophosphorylation events can:

The identification of these sites has been accomplished through in vitro kinase assays coupled with mass spectrometry. Specific autophosphorylation sites have been found in different regions of CDPK1, including the N-terminal extension, which has been determined to be important for PfCDPK1 activation .

Understanding these autophosphorylation sites provides insights into CDPK1 regulation and may aid in the development of inhibitors targeting specific regulatory mechanisms.

What are the recommended protocols for assessing CDPK1 kinase activity in vitro?

For assessing CDPK1 kinase activity in vitro, the following methodological approaches are recommended:

• Autophosphorylation assays: Set up kinase reactions with recombinant CDPK1 in the presence of calcium (typically 2 mM CaCl₂) and ATP. The activity can be measured through autothiophosphorylation of CDPK1. Control reactions should include EGTA (2 mM) without added calcium to confirm calcium dependency .

• Transphosphorylation assays: Use synthetic peptide substrates or physiological substrates to measure the kinase's ability to phosphorylate targets. Radioactive (³²P-labeled ATP) or non-radioactive methods (phospho-specific antibodies) can be employed to detect phosphorylation .

• Calcium dependency testing: Perform activity assays with varying calcium concentrations to determine EC₅₀ values for calcium activation. This can be correlated with fluorescence spectroscopy data to understand structural changes upon calcium binding .

• Inhibitor sensitivity assays: For evaluating the effect of inhibitors, pre-incubate CDPK1 with varying concentrations of the inhibitor before initiating the kinase reaction. This approach is useful for determining IC₅₀ values and structure-activity relationships .

When conducting these assays, it's important to include appropriate controls, such as:

• No-calcium controls (with EGTA)

• No-enzyme controls

• Heat-inactivated enzyme controls

• Known inhibitor controls

How can I generate and validate CDPK1 mutants for functional studies?

To generate and validate CDPK1 mutants for functional studies, the following comprehensive approach is recommended:

• Design of mutations:

  • Use structural models based on homologous proteins (e.g., TgCDPK1 for PfCDPK1) to identify critical residues .

  • Focus on functional domains such as the gatekeeper residue, calcium-binding EF-hands, or residues involved in interdomain interactions.

  • Consider conservation across species when selecting residues for mutation.

• Generation of recombinant mutant proteins:

  • Use site-directed mutagenesis on expression plasmids containing wild-type CDPK1.

  • Express mutant proteins in bacterial systems as GST-fusion proteins for easy purification.

  • Verify protein expression by SDS-PAGE and Western blotting with both anti-GST and anti-CDPK1 antibodies .

• Functional validation methods:

  • Compare biochemical properties of wild-type and mutant proteins using:

    • In vitro kinase assays to assess catalytic activity

    • Calcium-binding studies using fluorescence spectroscopy or isothermal titration calorimetry

    • Structural analyses via circular dichroism to detect changes in secondary structure

  • Evaluate inhibitor sensitivity profiles of mutant proteins compared to wild-type .

• Generation of parasite lines with mutant CDPK1:

  • Use CRISPR/Cas9 technology to introduce mutations into the endogenous CDPK1 locus.

  • Screen transfectants by PCR and sequencing to confirm successful mutation.

  • Phenotypically characterize mutant parasites for growth, invasion efficiency, and inhibitor sensitivity .

What approaches can be used to study CDPK1-substrate interactions?

Several complementary approaches can be employed to identify and characterize CDPK1-substrate interactions:

• In vitro phosphorylation assays:

  • Use recombinant CDPK1 to phosphorylate candidate substrates in vitro.

  • Detect phosphorylation using ³²P-labeled ATP or phospho-specific antibodies.

  • Analyze phosphorylated proteins by mass spectrometry to identify specific phosphorylation sites .

• Phosphoproteomic approaches:

  • Compare phosphoproteomes of wild-type and CDPK1-inhibited or CDPK1-mutant parasites.

  • Use stable isotope labeling (SILAC) or isobaric tagging (TMT/iTRAQ) for quantitative analysis.

  • Focus on phosphopeptides that decrease upon CDPK1 inhibition or mutation.

• Substrate recognition motif analysis:

  • Determine CDPK1 consensus phosphorylation motifs using peptide arrays or oriented peptide library screening.

  • Use bioinformatic approaches to predict potential substrates based on the presence of consensus motifs.

  • Validate predictions by targeted in vitro kinase assays.

• Protein-protein interaction studies:

  • Use co-immunoprecipitation followed by mass spectrometry to identify proteins interacting with CDPK1.

  • Employ yeast two-hybrid or proximity-labeling techniques (BioID/APEX) to detect transient interactions.

  • Confirm interactions by reciprocal co-immunoprecipitation or in vitro binding assays.

• Domain-specific interaction analysis:

  • Generate truncated versions of CDPK1 to identify domains involved in substrate recognition.

  • Use pull-down assays with individual domains to map interaction interfaces .

How do I reconcile contradictory in vitro versus in vivo results with CDPK1 inhibitors?

Contradictory results between in vitro enzyme assays and in vivo parasite experiments, such as those observed with BKI 1294 on wild-type versus T145M mutant CDPK1, require careful analysis and interpretation. Here's a systematic approach to reconcile such discrepancies:

• Consider compensatory mechanisms: As demonstrated with CDPK1 T145M parasites, reduced CDPK1 activity may be compensated by upregulation of other kinases (CDPK5, CDPK6) or increased dependence on alternative pathways (PKG-mediated) . These compensatory changes may alter the parasite's response to inhibitors in ways not predictable from in vitro enzyme assays.

• Examine pharmacokinetic/pharmacodynamic factors:

  • Inhibitor uptake, metabolism, and subcellular distribution may differ between in vitro and cellular systems.

  • Protein expression levels and post-translational modifications in parasites may differ from recombinant systems.

  • The presence of binding proteins or scaffolds in cells might affect inhibitor access to targets.

• Design validation experiments:

  • Use genetic approaches (CRISPR/Cas9) to confirm target engagement by introducing resistance mutations.

  • Employ chemical-genetic approaches with analog-sensitive kinases to validate on-target effects.

  • Analyze transcriptional and proteomic changes induced by inhibitor treatment to identify compensatory mechanisms .

• Implement dual-targeting strategies:

  • Based on the observation that CDPK1 T145M parasites were more sensitive to PKG inhibition, consider testing combination treatments targeting multiple kinases.

  • Design experiments to test synergistic effects between inhibitors of CDPK1 and related kinases identified as upregulated in resistant parasites .

What experimental design considerations are important when studying allosteric regulation of CDPK1?

When investigating the allosteric regulation of CDPK1, several experimental design considerations are crucial:

• Domain interaction studies:

  • The tethering of the calcium-activated domain (CAD) to the kinase domain (KD) is essential for CDPK1 activity. Studies with TgCDPK1 have shown that separating these domains by introducing protease cleavage sites abolishes kinase activity, and this activity cannot be restored by simply mixing the separated domains .

  • Design experiments that examine the interface between these domains and investigate specific residues involved in transmitting conformational changes.

• Calcium concentration controls:

  • Use precise calcium buffers to establish defined free calcium concentrations.

  • Include both calcium-free (EGTA) and calcium-saturated conditions in all experiments.

  • Consider the biphasic nature of calcium binding, which suggests heterogeneity in EF-hand affinities .

• Structural transition analysis:

  • Employ multiple biophysical techniques to monitor structural changes:

    • Circular dichroism to track secondary structure changes (α-helical content increases with calcium binding)

    • Fluorescence spectroscopy to detect tertiary structure alterations (quenching and red shift upon calcium binding)

    • ANS binding to monitor exposure of hydrophobic surfaces

  • Consider time-resolved measurements to capture transient conformational states.

• Mutational analysis approach:

  • Target residues that may play dual roles in stabilizing both active and inactive conformations.

  • Focus on residues at domain interfaces or in key regulatory elements like the αC-helix.

  • Consider mutations that might destabilize the inactive conformation, such as the L96P mutation observed in TgCDPK1, which affects the interaction between αC-helix and a short helical segment containing residues 197-201 .

How should I design experiments to investigate the role of CDPK1 in parasite biology?

To effectively investigate CDPK1's role in parasite biology, a multi-faceted experimental approach is recommended:

• Conditional expression/depletion systems:

  • Implement conditional knockdown approaches (tetracycline-regulatable, auxin-inducible degron, etc.) to control CDPK1 expression levels.

  • Use stage-specific promoters to regulate CDPK1 expression at different life cycle stages.

  • Create dominant-negative mutants by expressing catalytically inactive versions of CDPK1.

• Chemical genetic approaches:

  • Generate parasites expressing analog-sensitive CDPK1 (with modified gatekeeper residues) that can be specifically inhibited by bulky ATP analogs.

  • Use gatekeeper mutants (like T145M) that confer resistance to certain inhibitors to validate on-target effects .

  • Combine chemical inhibition with genetic approaches to achieve temporal control of CDPK1 function.

• Phenotypic analysis design:

  • Develop quantitative assays for parasite processes potentially regulated by CDPK1:

    • Host cell invasion (particularly for Toxoplasma gondii where CDPK1 is essential)

    • Egress from infected cells

    • Calcium signaling responses

    • Parasite motility

  • Implement time-lapse microscopy to capture dynamic processes.

  • Use flow cytometry-based approaches for high-throughput phenotypic analysis.

• Substrate identification strategy:

  • Conduct phosphoproteomic analysis comparing wild-type, CDPK1-inhibited, and CDPK1-depleted parasites.

  • Focus on phosphorylation events that correlate with CDPK1 activity levels.

  • Validate candidate substrates by in vitro kinase assays and mutagenesis of putative phosphorylation sites.

• Combinatorial targeting approach:

  • Based on findings that CDPK1-compromised parasites upregulate other kinases or become more dependent on PKG , design experiments combining inhibition/depletion of CDPK1 with manipulation of these compensatory pathways.

  • Test phenotypic consequences of simultaneously targeting multiple kinases.

  • Analyze transcriptional responses to CDPK1 manipulation to identify potential compensatory mechanisms.