Mechanism of Allosteric Inhibition of Plasmodium falciparum CGMP-Dependent Protein Kinase

Abstract

Malaria is a life-threatening disease responsible for about a million fatalities per year worldwide. Most of the malaria deaths are caused by Plasmodium falciparum. An essential regulator for the development of P. falciparum is the cyclic GMP (cGMP) dependent protein kinase (PfPKG). PfPKG is composed of a regulatory domain and a catalytic domain. In the absence of cGMP, the regulatory domain inhibits the kinase domain. Upon cGMP binding to the regulatory domain, the inhibition is released and PfPKG is activated. Targeting directly the active site of PfPKG poses a major selectivity challenge, since the kinase catalytic domains are highly conserved among eukaryotes. One approach to circumvent this problem is to selectively target less conserved allosteric sitesof PfPKG, such as the cGMP-binding domains (CBDs), which can be achieved through cGMP-analogs. Here, we report the mechanism of action for cGMP-antagonists of PfPKG to gain structural and dynamical insight on the otherwise elusive bound-inhibited state of PfPKG. This will provide another avenue to rationally design PfPKG-selective inhibitors for the treatment of malaria. NMR Chemical Shift Projection Analysis (CHESPA) of PfPKG CBD-D with cGMP-analogsshows that the cGMP-antagonist inhibits PfPKG not through a simple reversal of a two-state active-inactive equilibrium, but through multi-state equilibria sampling distinct holo-inactive intermediate that combine elements of both active and inactive states in different regions of CBD-D. This intermediate state exhibits inhibitory competent characteristics and also provides the inhibitors the avenue for preserving a high affinity for CBD-D. NMR spin relaxation measurements further complement the CHESPA data by probing the dynamical changes within the CBD-D of PfPKG that occur upon inhibition.