Disrupting the Allosteric Interaction between the Plasmodium falciparum cAMP-dependent Kinase and Its Regulatory Subunit

Dene R Littler,Hayley E Bullen,Katherine L Harvey,Travis Beddoe,Brendan S Crabb,Jamie Rossjohn,Paul R Gilson

doi:10.1074/jbc.m116.750174

Abstract

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

Highlights

The ubiquitous second messenger cyclic adenosine monophosphate (cAMP) mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites
Following purification of the PfPKA-R constructs, UV-visible light absorbance spectra indicated the presence of a 260-nm absorbance peak presumed to be bound nucleotide scavenged from the Escherichia coli lysate
This refolded apo-PfPKA-R.141.441 sample represents a conformation of PfPKA-R with two empty cyclic nucleotide-binding domains (CBDs)-binding sites primed for re-association with the protein kinase A (PKA)-C [22]

Summary

Introduction

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. The phylum Apicomplexa to which Plasmodium belongs is evolutionarily distant to most commonly studied organisms [4], and significant phylogenetic diversity separates the Plasmodium kinome from other systems studied This diversity can be exploited if inhibitory compounds can be developed that selectively block parasite regulatory enzymes while leaving orthologous human variants unaffected [5]. The regulatory PKA-R subunit is a highly dynamic molecular switch [16, 17] that serves as a competitive inhibitor of the catalytic subunit, DECEMBER 2, 2016 VOLUME 291 NUMBER 49

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