Publication Date
6-19-2020
Journal
Journal of Biological Chemistry
DOI
10.1074/jbc.RA120.013070
PMID
32317283
PMCID
PMC7307193
PubMedCentral® Posted Date
4-21-2020
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Allosteric Regulation, Binding Sites, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Plasmodium falciparum, Protein Domains, Protozoan Proteins, Recombinant Proteins, Surface Plasmon Resonance, cyclic GMP (cGMP), malaria, nuclear magnetic resonance (NMR), signaling, plasmodium, protein kinase G (PKG), 8-NBD-cGMP, CNB, cyclic nucleotide–binding domain (CBD), kinase signaling, PfPKG
Abstract
Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum. Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog–bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive “mixed” intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.
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