Pooja Kesari

Toxoplasma gondii , a worldwide prevalent parasite, is responsible for causing toxoplasmosis by infecting almost all warm-blooded animals, including humans. To establish a successful infection, the parasite exports a series of effector proteins which modulates the host immune system; Golgi-resident T. gondii aspartyl protease 5 ( Tg ASP5) plays an essential role in the maturation and export of these effector proteins. This is the first report of the detailed structural investigation of the Tg ASP5 mature enzyme. Molecular modeling and all-atom simulation provided in-depth knowledge of the active site architecture of Tg ASP5. The analysis of the binding mode of TEXEL substrate highlighted the importance of the active site residues forming the pocket; the Ser505, Ala776 and Tyr689 in the S2 binding pocket provide the specificity towards Arg at the P2 position. Our study also provides insights into the binding mode of the known inhibitor RRL _Statine. Screening the known aspartic protease inhibitors against Tg ASP5 active site and performing 100 ns all-atom molecular dynamic simulations, MM-PBSA binding energy calculations provided the best nine inhibitor protein complexes. Besides that, Principal Component Analysis (PCA) was employed to identify the change in protein dynamics with respect to the substrate and ligand binding. Tg ASP5 is essential for the fitness and virulence of the parasite; inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. Our study put forth the inhibitors which can act as initial scaffolds for developing potent mechanistic inhibitors against Tg ASP5.

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.

Plasmodium falciparum plasmepsin X ( Pf PMX), 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 Pf PMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to Pf PMX. The maturation of Pf PMX 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 Pf PMX. We present molecular details of inactivation, maturation, and inhibition of Pf PMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

The amino acid‐binding receptors, a component of ABC transporters, have evolved to cater to different specificities and functions. Of particular interest are cystine‐binding receptors, which have shown broad specificity. In the present study, a putative periplasmic cystine‐binding protein from Candidatus Liberibacter asiaticus (CLasTcyA) was characterized. Analysis of the CLasTcyA sequence and crystal structures in the ligand‐bound state revealed novel features of CLasTcyA in comparison to related proteins. One of the unique features found in CLasTcyA structure was the positioning of the C‐terminal extended loop of one chain very close to the substrate‐binding site of the adjacent monomer in the asymmetric unit. The presence of a disulphide bond, unique to Candidatus Liberibacter family, holds the C‐terminal extended loop in position. Analysis of the substrate‐binding pocket of CLasTcyA suggested a broad specificity and a completely different orientation of the bound substrates in comparison to related protein structures. The open conformation for one of the two chains of the asymmetric unit in the Arg‐bound structure revealed a limited open state (18.4°) for CLasTcyA as compared to open state of other related proteins (~ 60°). The strong interaction between Asp126 on helix‐α5 of small domain and Arg82 (bigger domain) restricts the degree of opening in ligand‐free open state. The dissociation constant of 1.26 μm by SPR and 3.7 μm by MST exhibited low affinity for the cystine. This is the first structural characterization of an l‐cystine ABC transporter from plant pathogen and our results suggest that CLasTcyA may have evolved to cater to its specific needs for its survival in the host.

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) ofEscherichia coliin batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

Auxin levels are tightly regulated within the plant cell, and its storage in the isolated cavity of proteins is a measure adopted by cells to maintain the availability of auxin. We report the first crystal structure of Wrightia tinctoria 11S globulin (WTG) in complex with Indole-3-acetic acid (IAA), an auxin, at 1.7 Å resolution. WTG hexamers assemble as a result of the stacking interaction between the hydrophobic surfaces of two trimers, leaving space for the binding of charged ligands. The bound auxin is stabilized by non-covalent interactions, contributed by four chains in each cavity. The presence of bound ligand was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-resolution mass spectrometry (HRMS). Here, we hypothesize that the cleavage of globulins by endopeptidases leads to the movement of the hydrophilic loop region from the surface to the periphery, leaving space for the binding of auxin, and promotes hexamer formation. As the process of germination proceeds, there is a change in the pH, which induces the dissociation of the hexamer and the release of auxin. The compact hexameric assembly ensures the long-term, stable storage of the hormone. This suggests a role for globulin as a novel player in auxin homeostasis.

2S albumin is a low-molecular-weight seed storage protein belonging to the prolamin superfamily. In the present work a small 2S albumin (WTA) protein of ~16 kDa has been purified from the seeds of Wrightia tinctoria. The WTA is a heterodimer protein with a small subunit of ~5 kDa and a larger subunit of ~11 kDa bridged together through disulphide bonds. The protein exhibits deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The protein also showed antibacterial activity against Morexalla catarrhalis. CD studies indicate a high α-helical content in the protein. The conserved disulphide bonds in the protein suggest that the WTA is highly stable under high pH and temperature like other 2S albumin.

Globulins are a major class of seed storage proteins which were thought to be enzymatically inactive. These proteins belong to the most ancient cupin superfamily. They can be graded into 11S legumin type and 7S vicilin type based on their sedimentation coefficients. Members from both classes share structural homology are thought to have evolved from either one-domain germin predecessor by duplication or by horizontal gene transfer of two-domain gene from bacteria to eukaryotes. Globulins are known to define the nutritional quality of the seeds, however, they are also involved in sucrose binding, desiccation, defense against microbes, hormone binding and oxidative stress etc. Major drawback with globulins is their tendency to bind to IgE. Studying structural-functional behavior of such protein can help in modifying proteins for enhanced functionality in food processing industries.