Profiles of global mutations in the human intercellular adhesion molecule-1 (ICAM-1) shed light on population-specific malaria susceptibility.

Plasmodium falciparum is responsible for malaria-related morbidity and mortality. PfEMP1 (P. falciparum erythrocyte membrane protein 1) mediates infected erythrocytes adhesion to various surface vascular receptors, including intercellular adhesion molecule-1 (ICAM-1), associating this interaction with severe malaria in several studies. Genetic variation in host ICAM-1 plays a significant role in determining susceptibility to malaria infection via clinical phenotypes such as the ICAM-1Kilifi variant which has been reported to be associated with susceptibility in populations. Our genomic and structural analysis of single nucleotide polymorphisms (SNPs) in ICAM-1 revealed 9 unique mutations each in its distinct A-type and BC-type PfEMP1 DBLß-interacting regions. These mutations are noted in only a few field isolates and mainly in the African/African American population. The ICAM-1Kilifi variant lies in a flexible loop proximal to the DBLß-interacting region. This analysis will assist in establishing functional correlations of reported global mutations via experimental and clinical studies and in the tailored design of population-specific genetic surveillance studies. Understanding host polymorphism as an evolutionary force in diverse populations can help to predict predisposition to disease severity and will contribute towards laying the framework for designing population-specific personalized medicines for severe malaria.

Single-Nucleotide Polymorphisms in Glucose-6-Phosphate Dehydrogenase and their Relevance for the Deployment of Primaquine as a Radical Cure for Malaria.

Malaria remains an important public health problem despite efforts to control it. Besides active transmission, relapsing malaria caused by dormant liver stages of Plasmodium vivax and Plasmodium ovale hypnozoites is a major hurdle in malaria control and elimination programs. Primaquine (PQ) is the most widely used drug for radical cure of malaria. Due to its anti-hypnozoite and gametocidal activity, PQ plays a key role in malaria relapse and transmission. The human enzyme glucose-6-phosphate dehydrogenase (G6PD) is crucial in determining the safety of PQ because G6PD-deficient individuals are prone to hemolysis if treated with PQ. Therefore, there is a need to study the prevalence of G6PD-deficient genetic variants in endemic populations to assess the risk of PQ treatment and the necessity to develop alternative treatments. In this work, we discuss the common G6PD variants, their varying enzymatic activity, and their distribution on the three-dimensional structure of G6PD. Our work highlights the important G6PD variants and the need for large-scale G6PD gene polymorphism studies to predict populations at risk of PQ-induced toxicity.

Structural and genomic analysis of single nucleotide polymorphisms in human host factor endothelial protein C receptor (EPCR) reveals complex interplay with malaria parasites.

Plasmodium parasites responsible for malaria follow a complex life cycle of which half takes place inside the human host. Parasites present diverse antigens at different stages of their life cycle and interact with many surface molecules to attach to and enter host cells. The CIDRα1 domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) in infected erythrocytes adheres to one such vascular receptor endothelial protein C receptor (EPCR). EPCR is implicated in the pathogenesis of severe malaria as preferential binding of CIDRα1 to endothelium results in widespread sequestration of infected erythrocytes leading to endothelium inflammation and severe disease. A single EPCR variant S219G is clinically reported to provide protection from severe malaria. In this work, we have collated all single nucleotide polymorphisms (SNPs) in EPCR from dbSNP. We structurally mapped the SNPs on the three-dimensional complex of EPCR and PfEMP1 CIDRα1. Analysis shows that most EPCR mutations lie on the receptor surface and are non-conservative. Of the 11 mutations in the CIDRα1-interaction region of EPCR, S88P, L96V/I, and R98L/H/P/C are seen with comparably higher occurrences in diverse populations. Our structural analysis details a framework of the interactions between the parasite ligand and host factor EPCR. These structural glimpses provide a blueprint for designing both field-based variant sequencing studies and vaccine development.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

Structural Profiles of SARS-CoV-2 Variants in India.

India was severely affected by several waves of SARS-CoV-2 infection that occurred during April-June 2021 (second wave) and December 2021-January 2022 (third wave) and thereafter, resulting in >10 million new infections and a significant number of deaths. Global Initiative on Sharing Avian Influenza Data database was used to collect the sequence information of ~10,000 SARS-CoV-2 patients from India and our sequence analysis identified three variants B.1.1.7 (alpha, α), B1.617.2 (delta, Δ), B.1.1.529 (Omicron, Oo) and one Omicron sub-variant BA.2.75 as the primary drivers for SARS-CoV-2 waves in India. Structural visualization and analysis of important mutations of alpha, delta, Omicron and its sub-variants of SARS-CoV-2 Receptor-Binding Domain (RBD) was performed and our analysis clearly shows that mutations occur throughout the RBD, including the RBD surface responsible for human angiotensin-converting enzyme 2 (hACE-2) receptor-binding. A comparison between alpha, delta and omicron variants/sub-variants reveals many omicron mutations in the hACE-2 binding site and several other mutations within 5 Å of this binding region. Further, computational analysis highlights the importance of electrostatic interactions in stabilizing RBD-hACE-2-binding, especially in the omicron variant. Our analysis explores the likely role of key alpha, delta and omicron mutations on binding with hACE-2. Taken together, our study provides novel structural insights into the implications of RBD mutations in alpha, delta and omicron and its sub-variants that were responsible for India's SARS-CoV-2 surge.

Genomic analysis of single nucleotide polymorphisms in malaria parasite drug targets.

Malaria is a life-threatening parasitic disease caused by members of the genus Plasmodium. The development and spread of drug-resistant strains of Plasmodium parasites represent a major challenge to malaria control and elimination programmes. Evaluating genetic polymorphism in a drug target improves our understanding of drug resistance and facilitates drug design. Approximately 450 and 19 whole-genome assemblies of Plasmodium falciparum and Plasmodium vivax, respectively, are currently available, and numerous sequence variations have been found due to the presence of single nucleotide polymorphism (SNP). In the study reported here, we analysed global SNPs in the malaria parasite aminoacyl-tRNA synthetases (aaRSs). Our analysis revealed 3182 unique SNPs in the 20 cytoplasmic P. falciparum aaRSs. Structural mapping of SNPs onto the three-dimensional inhibitor-bound complexes of the three advanced drug targets within aaRSs revealed a remarkably low mutation frequency in the crucial aminoacylation domains, low overall occurrence of mutations across samples and high conservation in drug/substrate binding regions. In contrast to aaRSs, dihydropteroate synthase (DHPS), also a malaria drug target, showed high occurrences of drug resistance-causing mutations. Our results show that it is pivotal to screen potent malaria drug targets against global SNP profiles to assess genetic variances to ensure success in designing drugs against validated targets and tackle drug resistance early on.

Prospects of halofuginone as an antiprotozoal drug scaffold.

Halofuginone is a clinically active derivative of febrifugine that was first isolated from the Chinese herb Dichroa febrifuga. The beneficial biological effects of halofuginone on various diseases including parasitic diseases, cancer, fibrosis, and autoimmune disorders have been investigated. Halofuginone has reduced toxic side effects when compared to febrifugine, an advantage that has led to the commercial availability of halofuginone-based antiparasitic drugs for animal use, and to human clinical trials for the treatment of tumors and fibrosis. This review summarizes advances in determining the mechanism of action of halofuginone, focusing on its antiparasitic role in malaria, cryptosporidiosis, coccidiosis, toxoplasmosis, and leishmaniasis. We discuss mechanistic insights into halofuginone's primary mode of action which involves inhibition of the prolyl-tRNA synthetase enzyme, which is crucial in protein synthesis. Halofuginone exemplifies the untapped wealth of plant-derived compounds in disease therapeutics.

Structural comparisons reveal diverse binding modes between nucleosome assembly proteins and histones.

Nucleosome assembly proteins (NAPs) are histone chaperones that play a central role in facilitating chromatin assembly/disassembly which is of fundamental importance for DNA replication, gene expression regulation, and progression through the cell cycle. In vitro, NAPs bind to the core histones H2A, H2B, H3, H4 and possibly to H1. The NAP family contains well-characterized and dedicated histone chaperone domain called the NAP domain, and the NAP-histone interactions are key to deciphering chromatin assembly. Our comparative structural analysis of the three three-dimensional structures of NAPs from S. cerevisiae, C. elegans, and A. thaliana in complex with the histone H2A-H2B dimer reveals distinct and diverse binding of NAPs with histones. The three NAPs employ distinct surfaces for recognizing the H2A-H2B dimer and vice versa. Though histones are highly conserved across species they display diverse footprints on NAPs. Our analysis indicates that understanding of NAPs and their interaction with histone H2A-H2B remains sparse. Due to divergent knowledge from the current structures analyzed here, investigations into the dynamic nature of NAP-histone interactions are warranted.

Structural insights into global mutations in the ligand-binding domain of VAR2CSA and its implications on placental malaria vaccine.

Placental malaria is a public health burden particularly in Africa as it causes severe symptoms and results in stillbirths or maternal deaths. Plasmodium falciparum protein VAR2CSA drives placental malaria (PM) in pregnant women by adhering to chondroitin sulfate A (CSA) on the placenta. VAR2CSA is a primary vaccine candidate for PM with two vaccines based on it already under clinical trials. The first cryo-EM three-dimensional structure of Pf CSA-VAR2CSA complex revealed crucial interacting residues considered to be highly conserved across P. falciparum strains. In the current study, we have conducted a global sequence analysis of 1,114 VAR2CSA field isolate sequences from more than nine countries across three continents revealing numerous mutations in CSA-binding residues. Further, structural mapping has revealed significant polymorphisms on the ligand binding surfaces. The variants from this limited set of 1,114 sequences highlight the concerns that are vital in current considerations for development of vaccines based on VAR2CSA for placental malaria.

Antigen exposure leads to rigidification of germline antibody combining site.

Immune complexes involving diverse antigens and corresponding antibodies were analyzed for mapping conformational transitions of an antibody before antigen binding, upon antigen binding and after antigen release. Molecular dynamics simulations of the two comprehensive datasets consisting of the antigen-free and antigen-bound structures of the germline antibodies 36-65 and BBE6.12H3 provided mechanistic model of antigen encounter by primary antibodies. While native germline antibodies exhibit substantial mobility in the antigen-combining sites, their antigen-bound states exhibit relatively rigid conformations, even in the absence of the antigen suggesting preservation of the structural state after antigen release. It is proposed that acquired rigidity by a germline antibody upon antigen binding may be the first step in affinity maturation in favor of that antigen.

Structure, localization and histone binding properties of nuclear-associated nucleosome assembly protein from Plasmodium falciparum.

BACKGROUND: Nucleosome assembly proteins (NAPs) are histone chaperones that are crucial for the shuttling and incorporation of histones into nucleosomes. NAPs participate in the assembly and disassembly of nucleosomes thus contributing to chromatin structure organization. The human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. METHODS: Three-dimensional crystal structure of PfNapS has been determined and analysed. Gene knockout and localization studies were also performed on PfNapS using transfection studies. Fluorescence spectroscopy was performed to identify histone-binding sites on PfNapS. Extensive sequence and structural comparisons were done with the crystal structures available for NAP/SET family of proteins. RESULTS: Crystal structure of PfNapS shares structural similarity with previous structures from NAP/SET family. Failed attempts to knock-out the gene for PfNapS from malaria parasite suggest essentiality in the parasite. GFP-fused PfNapS fusion protein targeting indicates cellular localization of PfNapS in the parasite nucleus. Fluorescence spectroscopy data suggest that PfNapS interacts with core histones (tetramer, octamer, H3, H4, H2A and H2B) at a different site from its interaction with linker histone H1. This analysis illustrates two regions on the PfNapS dimer as the possible sites for histone recognition. CONCLUSIONS: This work presents a thorough analysis of the structural, functional and regulatory attributes of PfNapS from P. falciparum with respect to previously studied histone chaperones.

Iodide-SAD, SIR and SIRAS phasing for structure solution of a nucleosome assembly protein.

The crystal structure of Plasmodium falciparum nucleosome assembly protein (PfNapL) was determined by iodide-SAD/SIRAS phasing methods using iodide-SAD data to 3.0 A resolution and native data to 2.4 A resolution. Halide-derivatized PfNapL crystals were obtained using the quick cryo-soaking method in which the native crystals were soaked in a cryosolution consisting of 500 mM NaI for a short period of 30-60 s and data were collected at an in-house X-ray source using Cu Kalpha radiation. Despite a low anomalous signal-to-noise ratio of <1.2 in the >3.5 A resolution bin, the data were sufficient to determine the structure by SAD/SIR/SIRAS methods using the soaked iodides. Previously, structure solution had failed with both molecular-replacement and selenomethionine-derivatization techniques owing to reasons that are detailed in this work. The phasing at low resolution with three iodides per monomer with high temperature factors was successful using any of the SAD, SIR or SIRAS methods.

Structural insights into chondroitin sulphate A binding Duffy-binding-like domains from Plasmodium falciparum: implications for intervention strategies against placental malaria.

BACKGROUND: Placental malaria is typified by selective clustering of Plasmodium falciparum in the intervillous blood spaces of the placenta. Sequestration of malaria parasite in the human placenta is mediated by interactions between chondroitin sulphate A (CSA) on the syncytiotrophoblasts and proteins expressed on the surface of infected human erythrocytes. Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) encoded by the var2CSA gene is believed to be the main parasite ligand for CSA-mediated placental binding. METHODS: Extensive sequence and structure comparisons of the various CSA-binding and non-binding DBL domains from the var2CSA gene from A4 and 3D7 strains of P. falciparum were performed. Three-dimensional structural models of various DBL domains were built and analysed with a view to assessing conservation of CSA interaction sites across various DBL domains. RESULTS: Each of the six DBL domains from var2CSA are likely to retain the disulfide linkages evident from previously published DBL domain crystal structures. The number of disulfide linkages between the various DBL domains analysed varies from three to seven, of which two are conserved across all DBL domains. The conserved disulfide linkages are distributed within the respective three sub-domains and only one linkage is shared by sub-domains I and II. Major differences between CSA-binding DBL domains are in the loop regions, which tie the alpha helices together, and in variable length terminal extensions. Intriguingly, a crucial loop from A4 DBL 3X which provides the important Gly and Lys residues that chelate the bound sulphate is missing or significantly altered in all other DBL domains that interact with CSA. Further analysis of the proposed sulphate and predicted CSA-binding site indicates either none or very low level of conservation among the critical interacting residues. CONCLUSION: Structural comparisons of the three-dimensional structures of CSA-binding DBL domains indicates that the proposed CSA interaction site on A4 DBL 3X is unlikely to be conserved across the other CSA-binding DBL domains from var2CSA. Therefore, the 4 CSA-binding DBL domains encoded by var2CSA are unlikely to have common architectures to their CSA recognition sites. These structural insights have clear implications in using CSA-binding DBL domains for vaccines against placental malaria as it is proposed that the various CSA-binding DBL domains on var2CSA will recognize their CSA ligands differently.

Crystal structure of malaria parasite nucleosome assembly protein: distinct modes of protein localization and histone recognition.

Nucleosome assembly proteins (NAPs) are histone chaperones that are essential for the transfer and incorporation of histones into nucleosomes. NAPs participate in assembly and disassembly of nucleosomes and in chromatin structure organization. Human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain structural insights into the mechanism of NAPs, we have determined and analyzed the crystal structure of PfNapL at 2.3 A resolution. PfNapL, an ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b (beta)/INHAT. The PfNapL monomer is comprised of domain I, containing a dimerization alpha-helix, and a domain II, composed of alpha-helices and a beta-subdomain. Structural comparisons reveal that the "accessory domain," which is inserted between the domain I and domain II in yeast NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression of green fluorescent protein-tagged PfNapL confirmed its exclusive localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene were not successful, indicating its essential role for the malaria parasite. A detailed analysis of PfNapL structure suggests unique histone binding properties. The crucial structural differences observed between parasite and yeast NAPs shed light on possible new modes of histone recognition by nucleosome assembly proteins.

Structure of a superoxide dismutase and implications for copper-ion chelation.

Superoxide dismutase (SOD) plays a central role in cellular defence against oxidative stress and is of pharmaceutical importance. The SOD from Potentilla atrosanguinea (Pa-SOD) is a unique enzyme as it possesses free-radical scavenging capability at temperatures ranging between 263 and 353 K. The crystal structure of recombinant Pa-SOD has been determined to 2.3 A resolution. The active-site residues are well ordered and additional water molecules are present in place of a bound copper ion. There is a significant difference in the relative orientation of the two subunits of Pa-SOD and asymmetry is also present in numerous hydrogen-bonding interactions. Structures of SODs, both bound with copper and unbound, have been compared with respect to the orientation of the electrostatic and Greek-key loops. This analysis provides new insights into the copper-chelation process in SODs. Several new structural features in Pa-SOD which may be responsible for its unique properties of thermostability and expanded range of antioxidant activity are also highlighted.

Crystal structure of soluble domain of malaria sporozoite protein UIS3 in complex with lipid.

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-A resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3(130-229)) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3(130-229) is a novel, compact, and all alpha-helical structure bound to one molecule of PE. The PfUIS3(130-229)-PE complex structure reveals a novel binding site with specific interactions between PfUIS3(130-229) and the PE head group. One acyl chain of PE wraps around part of PfUIS3(130-229) and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

SAD phasing of a structure based on cocrystallized iodides using an in-house Cu Kalpha X-ray source: effects of data redundancy and completeness on structure solution.

Superoxide dismutase (SOD) from Potentilla atrosanguinea (Wall. ex. Lehm.) was crystallized using 20% PEG 3350 and 0.2 M ammonium iodide and diffraction data were collected to 2.36 A resolution using an in-house Cu Kalpha X-ray source. Analyses show that data with a redundancy of 3.2 were sufficient to determine the structure by the SAD technique using the iodine anomalous signal. This redundancy is lower than that in previous cases in which protein structures were determined using iodines for phasing and in-house copper X-ray sources. Cocrystallization of proteins with halide salts such as ammonium iodide in combination with copper-anode X-ray radiation can therefore serve as a powerful and easy avenue for structure solution.