Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite
Ravi Jain,Pinki Dey,Sakshi Gupta,Soumya Pati,Arnab Bhattacherjee,Manoj Munde,Shailja Singh,Shailja Singh +7 more
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TLDR
Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target, setting a precedent for the rational design of 14- 3-3 based PPI inhibitors by utilizing 14-2-3 recognition motif peptides, as a potential antimalarial strategy.Abstract:
Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs, and confer conformational changes leading to modulation of functional parameters of their target proteins. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have annotated and characterized PF3D7_0818200 as 14-3-3 isoform I in Plasmodium falciparum 3D7, and its interaction with one of the key kinases of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Molecular dynamics simulation studies indicated that CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The 14-3-3I/CDPK1 interaction was validated in vitro with ELISA and SPR, which confirmed that the interaction is phosphorylation dependent, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: Mode I-type ARSHpSYPA and Mode II-type RLYHpSLPA, by simulation studies and ITC. This interaction was found to marginally enhance CDPK1 functional activity. Furthermore, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.read more
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References
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Role of the β isoform of 14-3-3 proteins in cellular proliferation and oncogenic transformation
TL;DR: Findings indicate that 14-3-3 beta has oncogenic potential, mainly through enhancement of Raf-1 activation and resultant augmentation of signaling in the MAPK cascade.
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Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion
Abhisheka Bansal,Shailja Singh,Kunal R. More,Dhiraj Hans,Kuldeep Nangalia,Manickam Yogavel,Amit Sharma,Chetan E. Chitnis +7 more
TL;DR: In this article, a peptide P3 from the junction domain of Plasmodium falciparum CDPK1 (PfCDPK) as well as purfalcamine inhibit functional activity to block microneme discharge and erythrocyte invasion.
Journal ArticleDOI
C-terminal Recognition by 14-3-3 Proteins for Surface Expression of Membrane Receptors
Brian Coblitz,Sojin Shikano,Meng Wu,Sandra B. Gabelli,Lisa M. Cockrell,Matt Spieker,Yoshiro Hanyu,Haian Fu,L. Mario Amzel,Min Li +9 more
TL;DR: The C-terminal SWTY motif, although different from mode I and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant (KD) of 0.17 μm, comparable with that of internal canonical binding peptides.
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PyTMs: a useful PyMOL plugin for modeling common post-translational modifications
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PfCDPK1 mediated signaling in erythrocytic stages of Plasmodium falciparum
Sudhir Kumar,Manish Kumar,Roseleen Ekka,Jeffrey D. Dvorin,Jeffrey D. Dvorin,Aditya S. Paul,Anil K. Madugundu,Tim W. Gilberger,Harsha Gowda,Manoj T. Duraisingh,T. S. Keshava Prasad,T. S. Keshava Prasad,Pushkar Sharma +12 more
TL;DR: The signaling network of PfCDPK1 is delineated and light is shed on mechanisms via which it regulates invasion through phosphoproteomics and conditional knockdown, which establishes that this kinase is critical for the invasion of host erythrocytes.