Abstract:
Malaria is a global health threat that causes 300 – 500 million clinical cases annually, resulting in approximately 2 million deaths. Chemotherapy and prophylaxis are becoming less effective because of increasing drug resistance by the parasite. Resurgence of malaria calls for the development of mechanistically novel drugs. The bifunctional organization of the two rate-limiting enzymes, AdoMetDC and ODC, in the P. Falciparum polyamine pathway and the presence of six parasite-specific inserts, present potential target sites for novel Plasmodia-specific drugs. The inserts are species-specific, hydrophilic, low complexity segments and form non-globular domains. The inserts are involved in intra- and interdomain interactions, which are important for stability and activity of the bifunctional construct. This study investigated properties of the parasite-specific inserts, one being the mobility of the O1 insert and the other the secondary structures present in the parasitespecific inserts. It is postulated that the mobility of the O1 insert plays a role in either heterotetrameric complex formation of the bifunctional construct or that the O1 insert acts as a “lid” to the ODC active site, which is necessary for catalytic function. Successful mutagenesis of the O1 flanking Gly residues to Ala, rendered the O1 insert immobile. The probable immobility of the O1 insert had a detrimental effect on the activity of both the AdoMetDC and ODC domains of the bifunctional protein. Molecular dynamics studies showed that movement restriction of the insert caused a conformational change in the ODC monomers. The decrease of both domain activities upon movement restriction of the O1 insert suggests that the insert is involved in protein-protein interactions, which is communicated throughout the protein. In addition, the roles of selected, predicted secondary structures in the Hinge, O1 and O2 parasite-specific inserts were investigated. á-Helices were disrupted by the introduction of a Pro residue, â-plates were removed with deletion mutagenesis. The effects of the secondary structure alterations on protein activity were monitored in the bifunctional PfAdoMetDC/ODC protein. Both domain activities were affected by the disruptions, although the ODC domain was more sensitive to the small changes. The results obtained in this study showed that the secondary structures in the parasite-specific insert are important for activity of both the AdoMetDC and ODC domains of the bifunctional protein, possibly via interdomain protein-protein interactions. The delineation of essential intra- and interdomain protein-protein interactions presents possible interaction sites for disruptive molecules in the combat against malaria. Copyright
