ABSTRACT
Plasmodium species are causative agents of malaria, a disease that is a serious global health concern. FDA-approved HIV-1 protease inhibitors (HIV-1 PIs) have been reported to be effective in reducing the infection by Plasmodium parasites in the population co-infected with both HIV-1 and malaria. However, the mechanism of HIV-1 PIs in mitigating Plasmodium pathogenesis during malaria/HIV-1 co-infection is not fully understood. In this study we demonstrate that HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum. Crystal structures of the complexes of PMII with both drugs have been determined. The inhibitors interact with PMII via multiple hydrogen bonding and hydrophobic interactions. The P4 moiety of RTV forms additional interactions compared to LPV and exhibits conformational flexibility in a large S4 pocket of PMII. Our study is also the first to report inhibition of P. falciparum PMX by RTV and the mode of binding of the drug to the PMX active site. Analysis of the crystal structures implies that PMs can accommodate bulkier groups of these inhibitors in their S4 binding pockets. Structurally similar active sites of different vacuolar and non-vacuolar PMs suggest the potential of HIV-1 PIs in targeting these enzymes with differential affinities. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing HIV-1 PIs as antimalarial drugs.
ABSTRACT
Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.
Subject(s)
Aspartic Acid Endopeptidases , Enzyme Precursors , Plasmodium falciparum , Protozoan Proteins , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Enzyme Precursors/chemistry , Plasmodium falciparum/enzymology , Protozoan Proteins/chemistryABSTRACT
Protein Crystallography or Macromolecular Crystallography (MX) started as a new discipline of science with the pioneering work on the determination of the protein crystal structures by John Kendrew in 1958 and Max Perutz in 1960. The incredible achievements in MX are attributed to the development of advanced tools, methodologies, and automation in every aspect of the structure determination process, which have reduced the time required for solving protein structures from years to a few days, as evident from the tens of thousands of crystal structures of macromolecules available in PDB. The advent of brilliant synchrotron sources, fast detectors, and novel sample delivery methods has shifted the paradigm from static structures to understanding the dynamic picture of macromolecules; further propelled by X-ray Free Electron Lasers (XFELs) that explore the femtosecond regime. The revival of the Laue diffraction has also enabled the understanding of macromolecules through time-resolved crystallography. In this review, we present some of the astonishing method-related and technological advancements that have contributed to the progress of MX. Even with the rapid evolution of several methods for structure determination, the developments in MX will keep this technique relevant and it will continue to play a pivotal role in gaining unprecedented atomic-level details as well as revealing the dynamics of biological macromolecules. With many exciting developments awaiting in the upcoming years, MX has the potential to contribute significantly to the growth of modern biology by unraveling the mechanisms of complex biological processes as well as impacting the area of drug designing.
Subject(s)
Proteins , Synchrotrons , Crystallography , Lasers , Macromolecular SubstancesABSTRACT
Plasmodium parasites that cause malaria produce plasmepsins (PMs), pepsin-like aspartic proteases that are important antimalarial drug targets due to their role in host hemoglobin degradation. The enzymes are synthesized as inactive zymogens (pro-PMs), and the mechanism of their conversion to the active, mature forms has not been clearly elucidated. Our structural investigations of vacuolar pro-PMs with truncated prosegment (pro-tPMs) reveal that the formation of the S-shaped dimer is their innate property. Further structural studies, biochemical analysis, and molecular dynamics simulations indicate that disruption of the Tyr-Asp loop (121p-4), coordinated with the movement of the loop L1 (237-247) and helix H2 (101p-113p), is responsible for the extension of the pro-mature region (harboring the cleavage site). Consequently, under acidic pH conditions, these structural changes result in the dissociation of the dimers to monomers and the protonation of the residues in the prosegment prompts its unfolding. Subsequently, we demonstrated that the active site of the monomeric pro-tPMs with the unfolded prosegment is accessible for peptide substrate binding; in contrast, the active site is blocked in folded prosegment form of pro-tPMs. Thus, we propose a novel mechanism of auto-activation of vacuolar pro-tPMs that under acidic conditions can form a catalytically competent active site. One monomer cleaves the prosegment of the other one through a trans-activation process, resulting in formation of mature enzyme. As a result, once a mature enzyme is generated, it leads to the complete conversion of all the inactive pro-tPMs to their mature form. DATABASE: Atomic coordinates and structure factors have been submitted in the Protein Data Bank (PDB) under the PDB IDs 6KUB, 6KUC, and 6KUD.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Plasmodium falciparum/enzymology , Amino Acid Sequence , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Catalytic Domain , Hydrogen-Ion Concentration , Models, Molecular , Protein Conformation , Sequence AlignmentABSTRACT
Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Ethylenediamines/chemistry , Isoquinolines/chemistry , Peptidomimetics/pharmacology , Thiazoles/chemistry , Antimalarials/chemistry , Antimalarials/pharmacology , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Crystallography, X-Ray , Drug Evaluation, Preclinical/methods , Ethylenediamines/pharmacology , Hemoglobins/metabolism , Humans , Isoquinolines/pharmacology , Molecular Targeted Therapy/methods , Peptidomimetics/chemistry , Plasmodium falciparum/drug effects , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Thiazoles/pharmacologyABSTRACT
The 2 µ plasmid of budding yeast shows high mitotic stability similar to that of chromosomes by using its self-encoded systems, namely partitioning and amplification. The partitioning system consists of the plasmid-borne proteins Rep1, Rep2 and a cis-acting locus STB that, along with several host factors, ensures efficient segregation of the plasmid. The plasmids show high stability as they presumably co-segregate with chromosomes through utilization of various host factors. To acquire these host factors, the plasmids are thought to localize to a certain sub-nuclear locale probably assisted by the motor protein, Kip1 and microtubules. Here, we show that the microtubule-associated proteins Bik1 and Bim1 are also important host factors in this process, perhaps by acting as an adapter between the plasmid and the motor and thus helping to anchor the plasmid to microtubules. Abrogation of Kip1 recruitment at STB in the absence of Bik1 argues for its function at STB upstream of Kip1. Consistent with this, both Bik1 and Bim1 associate with plasmids without any assistance from the Rep proteins. As observed earlier with other host factors, lack of Bik1 or Bim1 also causes a cohesion defect between sister plasmids leading to plasmid missegregation.