Malaria and Plasmodium falciparum PKG Activation
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Questions and Answers

What was the approximate number of malaria deaths in 2015?

  • 438,000 (correct)
  • 300,000
  • 500,000
  • 600,000

Which organism is responsible for transmitting malaria?

  • Male Anopheles mosquito
  • Female Aedes mosquito
  • Female Anopheles mosquito (correct)
  • Female Culex mosquito

Which variant of malaria is considered the most dangerous?

  • Plasmodium ovale
  • Plasmodium malariae
  • Plasmodium vivax
  • Plasmodium falciparum (correct)

What role does cGMP play in Plasmodium?

<p>Facilitates gametogenesis and exflagellation (B)</p> Signup and view all the answers

Which of the following is an effector of cGMP in Plasmodium?

<p>Pf PKG (C)</p> Signup and view all the answers

What is the primary role of cAMP signaling in Plasmodium?

<p>Controlling apical exocytosis and sporozoite motility (A)</p> Signup and view all the answers

Which stage of the malaria lifecycle is associated with schizogony?

<p>Asexual blood stage (C)</p> Signup and view all the answers

The distribution of malaria is most directly related to which factor?

<p>Climate and living area of the mosquito (C)</p> Signup and view all the answers

What was the activation constant (Kact) for the full length Pf PKG?

<p>330 ± 20 nM (D)</p> Signup and view all the answers

Which mutation was made to disrupt cGMP binding in CNB-B?

<p>R250K (C)</p> Signup and view all the answers

How did the specific activity of both mutant proteins compare to Pf PKG 158-853?

<p>Reduced to 50% (B)</p> Signup and view all the answers

What effect did the mutation of CNB-D (R492K) have on Pf PKG activation?

<p>Strongly influenced Pf PKG activation (B)</p> Signup and view all the answers

What range did the comparable affinities for cGMP of the mutant proteins display?

<p>70 to 110 nM (D)</p> Signup and view all the answers

Which peptide was tested on Pf PKG kinase activity?

<p>ERNKKKAIFSNDDF (D)</p> Signup and view all the answers

What are the residues that make up the capping triad of CNB-D?

<p>R484, Q532, D533 (B)</p> Signup and view all the answers

What method was used to assess Pf PKG kinase activity?

<p>Spectrophotometric assay (B)</p> Signup and view all the answers

What is the primary function of the catalytic domain in the Pf PKG encoded by the falciparum genome?

<p>Catalyzes cyclic nucleotide signaling (A)</p> Signup and view all the answers

How many cGMP binding domains are present in the regulatory domain of Pf PKG?

<p>Four (C)</p> Signup and view all the answers

Which antimalarial drug is not mentioned in the context of treatment challenges for malaria?

<p>Quinidine (B)</p> Signup and view all the answers

In what way does Pf PKG differ from its mammalian orthologue?

<p>Pf PKG has a larger size and lacks a dimerization domain (C)</p> Signup and view all the answers

What characteristic of P. falciparum’s life cycle complicates malaria treatment?

<p>It involves multiple life stages in both hosts (D)</p> Signup and view all the answers

Which binding domain is degenerate in the regulatory domain of Pf PKG?

<p>CNB-C (C)</p> Signup and view all the answers

What aspect of drug resistance significantly challenges malaria treatment?

<p>Development of next generation antimalarial drugs is essential (A)</p> Signup and view all the answers

What is the total molecular weight of Pf PKG as mentioned?

<p>97.5 kDa (D)</p> Signup and view all the answers

What effect did replacing the conserved Arg (R492) in CNB-D with a Lys residue have on cGMP binding?

<p>Decreased the binding affinity for cGMP (D)</p> Signup and view all the answers

Which of the following constructs showed the highest basal activity in the absence of cGMP?

<p>Pf PKG 401-853 (D)</p> Signup and view all the answers

What was the specific activity of Pf PKG 401-853 when activated by cGMP?

<p>5.3 U/mg (B)</p> Signup and view all the answers

What method was used to determine the active protein concentration for calculating specific activities?

<p>Inhibition by Compound 2 (B)</p> Signup and view all the answers

Which statement about the catalytic domain Pf PKG 519-853 is correct?

<p>It exhibited no detectable kinase activity with peptide substrates. (C)</p> Signup and view all the answers

Which CNB is described as a high-affinity, cGMP selective site?

<p>CNB-D (A)</p> Signup and view all the answers

What percentage of activity of the full-length protein does Pf PKG 401-853 achieve when activated by cGMP?

<p>60% (B)</p> Signup and view all the answers

What is the result of the expression of Pf PKG 158-853 and 275-853 in E. coli TP2000?

<p>Low basal activity with activation by cGMP (C)</p> Signup and view all the answers

What was a major consequence of TP2000 resulting in low yields?

<p>It limited the analysis of Pf PKG regulation. (C)</p> Signup and view all the answers

What is indicated about the regulatory domain of Pf PKG in comparison to hPKG?

<p>It shows significant differences that suggest it could be a drug target. (A)</p> Signup and view all the answers

At what concentrations of cGMP is the full-length Pf PKG protein activated?

<p>315 nM (B), 330 nM (D)</p> Signup and view all the answers

What was the result of the interaction analysis of FITC-labeled IS peptide with Pf PKG?

<p>The interaction was not demonstrated. (B)</p> Signup and view all the answers

Which part of the Pf PKG regulatory domain is crucial for its activation?

<p>cGMP binding. (C)</p> Signup and view all the answers

What characteristic of CNB-C of Pf PKG was established in previous studies?

<p>It binds neither cGMP nor cAMP. (B)</p> Signup and view all the answers

What was inferred about Pf PKG 32-853 mutant protein's activity?

<p>It can be activated by cGMP despite lacking the IS. (D)</p> Signup and view all the answers

What does the study suggest about the potential of Pf PKG as a drug target?

<p>It is a unique Plasmodium kinase with significant differences from hPKG. (B)</p> Signup and view all the answers

What is the effect of mutating the residues R209K and R333K in bovine PKA on cAMP affinity?

<p>It reduces cAMP affinity only by a factor of 10. (D)</p> Signup and view all the answers

How does the specific activity of Pf PKG cGMP deficient construct compare to Pf PKG 401-853?

<p>It is only slightly decreased. (A)</p> Signup and view all the answers

What is the main control mechanism for the catalytic activity of Pf PKG?

<p>Interactions involving the αC-helix. (C)</p> Signup and view all the answers

What happens when cGMP binds to Pf PKG?

<p>The αC-helix moves toward the PBC. (B)</p> Signup and view all the answers

What is indicated by the interaction between Y480 and R528 in Pf PKG?

<p>It stabilizes the αC-helix in the active state. (C)</p> Signup and view all the answers

Which CNB disruption resulted in a significant decrease (>70%) in activity?

<p>CNB-D (C)</p> Signup and view all the answers

What could be targeted for potential allosteric inhibitors in Pf PKG?

<p>The interaction between Y480 and R528. (D)</p> Signup and view all the answers

What effect did the addition of CNB-B and -C have on the activation constants?

<p>They had no change on the activation constants. (D)</p> Signup and view all the answers

What is the primary role of the N-terminal autoinhibitory sequence (IS) in Pf PKG?

<p>To inhibit catalytic activity (D)</p> Signup and view all the answers

Which cyclic nucleotide binding domain (CNB) is associated with high-affinity cGMP binding but is mentioned as degenerate?

<p>CNB-C (A)</p> Signup and view all the answers

Which construct demonstrates the highest basal activity in the absence of cGMP?

<p>Pf PKG 401-853 (B)</p> Signup and view all the answers

What type of cooperativity is observed with CNB-A and CNB-B in Pf PKG?

<p>Positive cooperativity (C)</p> Signup and view all the answers

What was the method used to determine the effect of cGMP on Pf PKG kinase activity?

<p>Microfluidic mobility-shift assay (MSA) (B)</p> Signup and view all the answers

What is described as the activation constant (Kact) in relation to Pf PKG?

<p>The concentration where half-maximal kinase activity is observed (A)</p> Signup and view all the answers

Which residue in CNB-A is highlighted as crucial for cGMP binding?

<p>R132 (C)</p> Signup and view all the answers

How does the specific activity of a cGMP deficient construct compare to Pf PKG 401-853?

<p>It is significantly lower in the presence of cGMP (C)</p> Signup and view all the answers

What was the activation constant (Kact) for the Pf PKG 32-853 mutant protein when activated by cGMP?

<p>315 nM (C)</p> Signup and view all the answers

Which part of Pf PKG is necessary for its activation by cGMP?

<p>Regulatory domain (C)</p> Signup and view all the answers

What significant property of CNB-C in Pf PKG was established in earlier studies?

<p>It does not bind either cGMP or cAMP. (C)</p> Signup and view all the answers

What mechanism is indicated for the autoinhibition of Pf PKG?

<p>Interaction of CNB-D without cGMP present. (A)</p> Signup and view all the answers

What was the observed effect of the R492K mutation on the cGMP binding affinity of the Pf PKG?

<p>It decreased cGMP binding affinity significantly. (A)</p> Signup and view all the answers

Which Pf PKG construct expressed in E. coli showed low basal activity in the absence of cGMP?

<p>Pf PKG 401-853 (D)</p> Signup and view all the answers

What outcome was observed regarding the interaction of a FITC-labeled IS peptide with Pf PKG?

<p>No interaction was demonstrated. (D)</p> Signup and view all the answers

What conclusion can be drawn regarding the regulatory domain of Pf PKG compared to hPKG?

<p>Pf PKG's regulatory domain differs significantly. (D)</p> Signup and view all the answers

What effect does the addition of cGMP have on the Pf PKG protein's activity?

<p>Activates the catalytic domain. (B)</p> Signup and view all the answers

What is the role of the conserved Arg (R492) in CNB-D of Pf PKG?

<p>It is critical for cGMP interaction. (B)</p> Signup and view all the answers

Which construct yielded no detectable kinase activity with PKStide or Kemptide?

<p>Pf PKG 519-853 (B)</p> Signup and view all the answers

What was indicated about the specific activity of Pf PKG 401-853 when activated by cGMP?

<p>It showed enhanced activity. (C)</p> Signup and view all the answers

Which cGMP binding domain is classified as low affinity and non-selective for cAMP and cGMP?

<p>CNB-B (C)</p> Signup and view all the answers

What percentage of activity does Pf PKG 401-853 achieve compared to the full-length protein when activated?

<p>60% (B)</p> Signup and view all the answers

What was noted about the specific activities determined by the Bradford assay?

<p>50-80% of protein concentration was catalytically active. (D)</p> Signup and view all the answers

What was the EC50 value for the cGMP binding of recombinant mutant protein Pf PKG 401-853 R492K?

<blockquote> <p>100,000 nM (C)</p> </blockquote> Signup and view all the answers

Which mutant proteins showed increased Kact values for cGMP?

<p>Pf PKG 401-853 R492K (D)</p> Signup and view all the answers

What change was observed in the Hill slopes when comparing Pf PKG 401-853 and full-length Pf PKG?

<p>Increased from 0.7 to 1.6 (A)</p> Signup and view all the answers

What was the specific activity of Pf PKG 401-853 when activated by cGMP compared to its mutants?

<p>Increased significantly (B)</p> Signup and view all the answers

Which method was used to determine the EC50 values for cGMP binding for the different constructs?

<p>Solution competition assay (B)</p> Signup and view all the answers

What does the term 'Kact' refer to in the context of cGMP activation?

<p>An indicator of catalytic efficiency (A)</p> Signup and view all the answers

What is indicated by the specific activities of the cGMP deficient constructs compared to Pf PKG 401-853?

<p>They show lower activities than Pf PKG 401-853 (D)</p> Signup and view all the answers

Which construct displayed the highest basal activity in the absence of cGMP?

<p>Pf PKG 401-853FP (C)</p> Signup and view all the answers

What impact does the binding of cGMP to CNB-D have on the Pf PKG protein?

<p>It induces a conformational change and stabilizes the active state. (D)</p> Signup and view all the answers

Which amino acid is involved in stabilizing the αC-helix when cGMP is not bound?

<p>Arginine (R528) (C)</p> Signup and view all the answers

What was the purpose of introducing the mutations Y480F, R528K, and D597N?

<p>To investigate the activation mechanism of Pf PKG. (B)</p> Signup and view all the answers

At what temperature and OD600 nm was expression of Pf PKG proteins induced in E. coli TP2000?

<p>37 °C, OD600 nm of 0.6−0.8 (A)</p> Signup and view all the answers

What is the purpose of using SDS-PAGE in protein analysis?

<p>To verify the purity of protein samples. (D)</p> Signup and view all the answers

What role does the CNB-D play in the activation of Pf PKG?

<p>It facilitates the binding of cGMP and induces conformational changes. (D)</p> Signup and view all the answers

What component was included in the elution buffer for His-tagged proteins?

<p>250 mM imidazole (C)</p> Signup and view all the answers

What was the purpose of using a French press in the procedure?

<p>To lyse the cells and release the protein content. (C)</p> Signup and view all the answers

What solvent was used to equilibrate the HiLoad 16/60 Superdex 75 gel filtration column?

<p>20 mM MOPS (pH 7.0) (B)</p> Signup and view all the answers

How does the introduction of a Lys at position 528 (R528K) affect Pf PKG?

<p>It disrupts the interaction with D597, affecting activation. (A)</p> Signup and view all the answers

Which method was used to calculate Kact values?

<p>Sigmoidal dose-response curves (C)</p> Signup and view all the answers

What is indicated by the interaction between residues Y480 and R528 in Pf PKG?

<p>It contributes to stabilizing the active form upon cGMP binding. (A)</p> Signup and view all the answers

What role does 2-mercaptoethanol serve in the buffers used?

<p>Stabilizes protein structure. (A)</p> Signup and view all the answers

What components were included in the reaction buffer for the fluorescence polarization assay?

<p>1 mM ATP, 220 μM NADH (D)</p> Signup and view all the answers

What was suggested by the 3-fold lower Kact value for the His-tagged protein constructs?

<p>The αC-helix cannot be stabilized longer in the inactive state. (C)</p> Signup and view all the answers

What does the Bradford assay measure?

<p>The concentration of proteins in samples. (D)</p> Signup and view all the answers

Flashcards

Malaria deaths in 2015

Approximately 438,000 people died from malaria in 2015.

Malaria Cause

Malaria is caused by Plasmodium protozoans.

Malaria Transmission

Malaria is transmitted by female Anopheles mosquitoes.

Malaria Distribution

Malaria distribution is linked to mosquito habitats.

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Dangerous Malaria Variant

Malaria tropica (caused by Plasmodium falciparum) is the most dangerous type.

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cGMP Role in Plasmodium

cGMP signaling is crucial for exflagellation and gametogenesis in Plasmodium's blood stage.

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cAMP Role in Plasmodium

cAMP signaling controls sporozoite motility and apical exocytosis in Plasmodium.

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Pf PKG

Pf PKG is the primary effector of cGMP in Plasmodium.

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Pf PKG isoform

A single protein kinase encoded by the Plasmodium falciparum genome, with regulatory and catalytic domains.

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Pf PKG Domains

Pf PKG has N-terminal regulatory and C-terminal catalytic domains, plus multiple cGMP binding domains.

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cGMP binding domains (CNBs)

Four cGMP binding parts (CNB-A/B/C/D) in Pf PKG's regulatory section. CNB-C is a bit unusual.

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Mammalian PKG vs Pf PKG

Mammalian PKGs have significantly different regulatory domains (fewer cGMP binding parts) and lack a dimerization domain compared to the larger Pf PKG.

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Malaria Life Cycle

Malaria parasites go through multiple stages in humans and mosquitoes, with controlled intracellular processes (including cyclic nucleotides and kinases).

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Drug Resistance

Current antimalaria drugs (e.g., chloroquine, atovaquone, etc.) are facing resistance from malaria organisms.

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Anti-malarial Drug Development

New antimalaria drugs are urgently needed to combat the threat from drug-resistant malaria strains.

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Cyclic Nucleotide Signaling

A process that's part of the regulated, intracellular processes of the malaria parasite life cycle.

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What is Pf PKG?

Pf PKG is a protein kinase in Plasmodium responsible for responding to cGMP signaling. It plays a key role in parasite development and survival.

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How is Pf PKG activated?

Pf PKG is activated by cGMP binding to its CNB domains, particularly CNB-D, which triggers a conformational change leading to kinase activity.

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What is the role of CNB-B and CNB-D in Pf PKG activation?

CNB-B and CNB-D are cyclic nucleotide binding domains in Pf PKG. Both bind cGMP, but CNB-D is crucial for Pf PKG activation. Mutations in CNB-D significantly reduce activation.

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Does Pf PKG activation require an N-terminal IS?

No, Pf PKG activation does not rely on an N-terminal inhibitory sequence (IS) interaction with the catalytic domain, unlike human PKG.

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What happens when cGMP binding to CNB-B is disrupted?

Disrupting cGMP binding to CNB-B (by mutating R250K) doesn't affect the activation constant of Pf PKG.

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What happens when cGMP binding to CNB-D is disrupted?

Disrupting cGMP binding to CNB-D (by mutating R492K) significantly impacts Pf PKG activation, increasing the activation constant.

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What is the significance of the CNB-D capping triad?

The capping triad in CNB-D (R484, Q532, D533) is critical for the proper structure and function of CNB-D, which in turn affects Pf PKG activation.

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What is the importance of studying Pf PKG?

Understanding Pf PKG regulation is essential for developing new anti-malaria drugs that target cGMP signaling pathways in Plasmodium.

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What is the purpose of studying Pf PKG?

Pf PKG, a Plasmodium kinase, is a potential drug target for treating malaria. Understanding its regulation is vital for developing effective therapies.

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How does Pf PKG differ from hPKG?

The regulatory domain of Pf PKG is structurally distinct from its human counterpart (hPKG).

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What is the role of the IS motif?

The IS motif is a region in Pf PKG that interacts with the catalytic domain, potentially regulating its activity.

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How does cGMP impact Pf PKG?

cGMP binding to Pf PKG activates it. However, the IS motif's role in this activation remains unclear.

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What happens to Pf PKG without the IS?

Pf PKG (32-853), missing the IS motif, still exhibits cGMP-dependent activation, suggesting an alternative activation mechanism.

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Is the CNB-C of Pf PKG functional?

Unlike hPKG, the CNB-C of Pf PKG is degenerate and doesn't bind cGMP or cAMP.

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Which part of Pf PKG is crucial for autoinhibition?

The CNB-D domain of Pf PKG is responsible for autoinhibition, preventing uncontrolled activation.

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Why is cGMP binding to CNB-D important?

cGMP binding to CNB-D is essential for Pf PKG activation. It likely disrupts the autoinhibitory mechanism.

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Pf PKG Activation

The activation of Pf PKG, a key kinase in the malaria parasite, is controlled by the αC-helix within its CNB-D domain. This helix can adopt two conformations: one inactive, bound to a catalytic domain, and one active, bound to a PBC and stabilized by interactions with other residues.

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Pf PKG CNB-D Role

The CNB-D domain of Pf PKG plays a central role in its activity. It contains the αC-helix, which changes conformation upon cGMP binding, switching between inactive and active Pf PKG states. The interaction between Y480 and R528 stabilizes the active conformation.

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cGMP Binding & Activation

When cGMP binds to Pf PKG, it causes a conformational change in the αC-helix within CNB-D. The helix moves towards the PBC, and this shift stabilizes the active state. Disrupting the interaction between Y480 and R528 prevents activation.

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Pf PKG Inactive State

In its inactive form, the αC-helix in CNB-D interacts with D597, stabilizing the catalytic domain in a state where it cannot phosphorylate substrates. The αC-helix prevents the activation of Pf PKG.

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Pf PKG Active State

When cGMP binds, the αC-helix re-orients, moving toward the PBC and interacting with Y480 and R528. This interaction stabilizes the active conformation, allowing Pf PKG to phosphorylate its substrates.

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Allosteric Inhibition Target

The interaction between Y480 and R528 is crucial for Pf PKG activation. Disrupting this interaction using allosteric inhibitors could be a novel strategy for fighting malaria. These inhibitors could include small molecules, peptides, or cyclic nucleotide analogues.

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Importance of CNBs

Pf PKG has multiple CNBs, which are essential for its full activity. While CNB-D is the primary regulator, other CNBs contribute to the overall activity. Disrupting CNBs can significantly reduce Pf PKG's activity.

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Comparison to Mammalian PKG

Unlike Pf PKG, mammalian PKGs have fewer cGMP binding domains and lack a dimerization domain. This difference in structure highlights the unique features of Pf PKG and provides a pathway for developing selective inhibitors.

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Pf PKG role

Pf PKG is a protein kinase crucial for the development and survival of the malaria parasite. It responds to cGMP signaling, a key process in the parasite's life cycle.

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What are CNBs?

CNBs are cyclic nucleotide binding domains found in Pf PKG. They are responsible for sensing cGMP levels and controlling the kinase's activation.

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CNB-D's role in activation

CNB-D is a high-affinity cGMP binding domain in Pf PKG. It's essential for activating Pf PKG. Disrupting cGMP binding to CNB-D significantly reduces activation.

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Pf PKG activation mechanism

cGMP binding to CNB-D triggers a conformational change in a region called the αC-helix in CNB-D. This change shifts the helix to an active state, allowing Pf PKG to phosphorylate substrates.

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Importance of Y480-R528 interaction

The interaction between Y480 and R528 is critical for stabilizing the active conformation of Pf PKG. Disrupting this interaction prevents activation.

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Allosteric inhibitors for Pf PKG

Allosteric inhibitors target the interaction between Y480 and R528, disrupting the activation of Pf PKG. These inhibitors can be small molecules, peptides, or cyclic nucleotide analogs.

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CNB-D

A cyclic nucleotide binding domain within Pf PKG. It binds cGMP with high affinity and is essential for activating the kinase.

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αC-Helix

A region in Pf PKG's CNB-D domain that undergoes a conformational change upon cGMP binding. This change is crucial for activating the kinase.

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Y480-R528 Interaction

An interaction between two amino acids in Pf PKG's CNB-D domain that stabilizes the active conformation of the kinase.

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Allosteric Inhibitors

Drugs that target the interaction between Y480 and R528 in Pf PKG, blocking the kinase's activation. This prevents the parasite from growing and surviving.

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How Does Pf PKG get Activated?

Pf PKG is activated when cGMP binds to its CNB-D domain, specifically in the αC-helix. This causes a conformational change in the protein, allowing Pf PKG to phosphorylate its targets.

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What Happens to Pf PKG without CNB-D?

Without CNB-D, Pf PKG cannot bind cGMP effectively, making it difficult to activate. This effectively stops the kinase from functioning, hindering the parasite's development.

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How does the structure of Pf PKG differ from Mammalian PKG?

Pf PKG has a more complex regulatory domain with multiple cGMP binding domains, including CNB-D, which is crucial for its activation. Mammalian PKG lacks this complexity and utilizes fewer cGMP binding domains.

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Why Study Pf PKG?

Pf PKG is a protein kinase essential for the survival and development of the malaria parasite. By understanding its regulation, scientists can develop new anti-malaria drugs that target cGMP signaling pathways, potentially leading to more effective treatments.

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Why is Y480-R528 interaction important?

The interaction between amino acids Y480 and R528 in Pf PKG's CNB-D domain stabilizes the active conformation of the kinase. This interaction is essential for full activation of Pf PKG.

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What are allosteric inhibitors?

Drugs that target specific regions in Pf PKG, such as the interaction between Y480 and R528, to prevent the activation of the kinase. They block the parasite's growth by interfering with Pf PKG's function.

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How does Pf PKG activation relate to the parasite's life cycle?

The activation of Pf PKG through cGMP binding to CNB-D is essential for key stages in the parasite's life cycle, such as its growth and development within red blood cells.

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What makes Pf PKG a potential drug target?

The unique structure of Pf PKG, particularly the CNB-D domain and the αC-helix, presents opportunities for the development of highly specific inhibitors that target the activation process, disrupting the parasite's growth.

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How does cGMP activate Pf PKG?

cGMP binds to the CNB-D domain of Pf PKG, specifically in the αC-helix. This triggers a conformational change, moving the helix to an active conformation.

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What is the role of CNB-D in Pf PKG activation?

CNB-D is a crucial cyclic nucleotide binding domain in Pf PKG. It binds cGMP with high affinity and is essential for activating the kinase.

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Why is the Y480-R528 interaction important?

The interaction between amino acids Y480 and R528 helps stabilize the active conformation of Pf PKG. Disrupting this interaction prevents activation.

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How do allosteric inhibitors target Pf PKG?

Allosteric inhibitors target the interaction between Y480 and R528 in Pf PKG, blocking the kinase's activation. This prevents the parasite from growing and surviving.

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What is the structure of Pf PKG's regulatory domain?

Pf PKG has a complex regulatory domain with multiple cGMP binding domains, including CNB-D. This is different from mammalian PKGs, which have fewer cGMP binding domains.

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Why is studying Pf PKG important?

Pf PKG is a promising target for anti-malaria drugs. By understanding its regulation, scientists can develop new therapies that effectively inhibit the parasite's growth.

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What makes Pf PKG a good target for anti-malaria drugs?

Pf PKG is essential for the malaria parasite's survival. Blocking its activity with drugs can disrupt the parasite's lifecycle and potentially lead to effective treatments.

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Study Notes

Plasmodium falciparum PKG Activation

  • PfPKG, a cGMP-dependent protein kinase, is crucial for malaria parasite proliferation (sexual and asexual)
  • PfPKG differs significantly from human PKG (hPKG) in domain organization
  • CNB-D (Cyclic Nucleotide Binding Domain D) plays a critical role in PfPKG activation and regulation unlike hPKG which is regulated by a pseudosubstrate autoinhibitory sequence (IS)
  • cGMP binding to CNB-D induces a conformational change, specifically involving the aC-helix
  • The inactive state is characterized by an interaction between Asp597 (catalytic domain) and Arg528 (aC-helix)
  • Arg528 also stabilizes cGMP binding by interacting with Tyr480 (phosphate binding cassette) in the active state
  • This unique activation mechanism of PfPKG, distinct from hPKG, suggests it as a potential drug target for malaria.

Malaria Disease Overview

  • Malaria is a major global infectious disease caused by Plasmodium parasites
  • Transmitted by female Anopheles mosquitoes
  • 214 million cases per year, with 438,000 deaths in 2015
  • Drug resistance is a significant challenge in malaria treatment.

cGMP Signaling in Malaria

  • cGMP signaling plays a crucial role in the asexual blood stage, exflagellation, gametogenesis, and schizogony in liver stages of malaria.
  • cAMP signaling controls events like apical exocytosis, sporozoite motility, anion transport, and RBC and liver cell invasion.
  • Plasmodium falciparum has a single PKG isoform, larger than mammalian orthologs, lacking a dimerization domain
  • The multiple CNBs (cyclic nucleotide binding domains) in the regulatory domain of PfPKG differ significantly from mammalian PKGs.

PfPKG Structure and Function

  • PfPKG consists of an N-terminal regulatory domain and a C-terminal catalytic domain.
  • The regulatory domains contain 4 CNBs, with CNB-C being degenerate (not functional)
  • PfPKG is larger than its mammalian orthologue with about 97.5 kDa
  • A recent study identified 107 phosphorylation sites on 69 proteins as cellular targets of PfPKG. These are involved in cell signalling, gene regulation, proteolysis, and transport.

Mechanism of PfPKG activation

  • The activation of PfPKG does not depend on the N-terminal IS motif interacting with the catalytic domain as in hPKG.
  • Deleting specific CNBs (such as CNB-D) in PfPKG creates constructs that still show activation by cGMP
  • The isolated CNB-D has high-affinity cGMP binding.
  • The amino acid arginine within the phosphate binding cassette (PBC), specifically Arg492 in CNB-D, is critical for cGMP binding and subsequent activation. Mutations affecting this residue show significant reduction in activity.
  • The C-helix of CNB-D plays a crucial role in PfPKG activation; its interaction with the catalytic domain is crucial for regulated activity. Mutations in the relevant amino acids (R528) significantly reduce activation.

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cGMP Binding Domain D Mediates a Unique Activation Mechanism PDF

Description

This quiz explores the role of PfPKG, a crucial protein kinase in the proliferation of malaria parasites. It discusses its unique activation mechanism in contrast to human PKG, highlighting its potential as a drug target. Gain insights into the molecular dynamics of malaria and the distinctive aspects of Plasmodium falciparum biology.

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