Biology subjects

Bhaumik, P.

Publications and source records attributed to Bhaumik, P..

17 recordsLinked to original sources

bioRxiv2026

Molecular insights into the regulation of coenzyme selection in NADP-specific glutamate dehydrogenase from Saccharomyces cerevisiae

Coenzyme specificity is one of the crucial aspects of the functional regulations in enzymes. Understanding the molecular basis of coenzyme specificity in glutamate dehydrogenase (GDH) is highly essential as this is the only enzyme that fulfils various physiological demands in terms of energy production and/or nitrogen assimilation via its strict functional regulation by connecting carbon and nitrogen metabolic cycles in every living organism. We have studied S. cerevisiae GDH3 (ScGDH3), as a model system for NADP-specific fungal GDHs. K272R and S250E mutants of ScGDH3 exhibited remarkable alteration in coenzyme specificity, with 15-55-fold and 3-14-fold more preference towards non-specific coenzymes [NAD(H)], respectively. S250E exhibited higher NADH affinity (~ 1.3-fold) than that of towards NADPH. K272R showed ~ 10-fold increased coenzyme affinity (1/KM) and binding affinity (1/kd) towards NADH, compared to the wild type enzyme (ScGDH3-WT). We solved the crystal structure of ScGDH3-K272R complexed with NADP+ at 2.50 [A] resolution with a unique oligomeric arrangement having two distinct subunit conformations (''open'' and ''super-closed'') and it is the first structure of a GDH from S. cerevisiae. Molecular dynamics (MD) simulations provide insights into NADH binding and its increased affinity to ScGDH3-K272R. Further structure-based sequence analyses unravelled that a lysine and an arginine are naturally preferred residues for providing NADP(H) and NAD(H) specificity, respectively for various amino acid dehydrogenases. Taken together biochemical and structural data, our study successfully illustrates the evolutionary conserved mechanism of coenzyme related regulation in fungal and other GDHs; as well as amino acid dehydrogenases.

biochemistry↗

bioRxiv2026

Conformational activation of GSK3β by an environmental toxicant suppresses hedgehog signalling

Primary cilium-dependent hedgehog signalling is essential for embryonic development, tissue patterning, and organ homeostasis, and its dysfunction causes ciliopathies, a clinically diverse spectrum of developmental and reproductive disorders. Whether environmental chemicals can phenocopy genetic ciliopathies by directly targeting ciliary kinase machinery has remained unknown. Here we show that endosulfan, a banned organochlorine pesticide linked to congenital and reproductive defects, suppresses hedgehog signalling by driving proteolytic processing of GLI transcription factors into repressor forms. Excluding ciliary receptor trafficking, cAMP signalling, and GLI-DNA binding, we identify PKA and GSK3{beta} as direct endosulfan targets: endosulfan allosterically fine-tunes PKA activity and, to our knowledge, is the first reported small-molecule activator of GSK3{beta}, stabilising its active conformation, a profile distinct from all known inhibitors. We further identify Cetn3 and Cep250 as novel GLI-regulated genes required for centriole cohesion, both of which are repressed upon endosulfan exposure, linking this kinase axis to the reproductive defects reported in exposed human populations and animal models. These findings establish a chemical-biological axis through which an environmental toxicant hijacks core kinase signalling to phenocopy a genetic ciliopathy.

cell biology↗

bioRxiv2026

Endosulfan rewires PKA and GSK3β to disrupt primary cilia-dependent Hedgehog signalling

Primary cilium-dependent Hedgehog signalling is essential for embryonic development, tissue patterning, and organ homeostasis, and its disruption causes a spectrum of developmental disorders collectively termed ciliopathies. Whether environmental toxicants can chemically induce ciliopathy-like states by targeting this pathway, however, remains poorly understood. Here we show that endosulfan, a banned organochlorine pesticide epidemiologically linked to severe congenital and reproductive defects in exposed human populations, disrupts Hedgehog signalling by driving GLI transcription factor processing into repressor forms and suppressing target gene expression at both transcriptional and protein levels. Having excluded direct effects on core ciliary receptors and GLI-DNA binding, we identify the pathway kinases PKA and GSK3{beta} as direct targets of endosulfan: endosulfan increases PKA activity through allosteric fine-tuning, and -- in a pharmacologically rare finding -- acts as the first reported small-molecule activator of GSK3{beta}, shifting the kinase toward a catalytically active conformation. We further identify Cetn3 and Cep250 as novel GLI-regulated genes required for centriole cohesion, both of which are repressed upon endosulfan exposure, providing a mechanistic link to the reproductive defects reported in exposed populations and animal models. These findings identify endosulfan as a candidate chemical inducer of ciliopathy and reveal how an environmental toxicant can hijack core kinase signalling to disrupt Hedgehog-dependent development.

cell biology↗

bioRxiv2026

Probing the role of residues lining the active site in the generation of glucose-tolerant variants of a fungal GH1 enzyme

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

biochemistry↗

bioRxiv2025

Deciphering Membrane Pore Formation Mechanisms of Plasmodium falciparum Perforin-Like Protein 1 (PfPLP1)

Membrane Attack Complex/Perforin (MACPF) domain proteins are {beta}-pore forming toxins ({beta}-PFTs) involved in the pathogenesis of various organisms. Among them, Perforin-like proteins (PLPs), produced by Plasmodium species, play essential roles in parasite invasion and egress. Due to increasing drug resistance in Plasmodium, PLPs represent promising but underexplored therapeutic targets, largely due to the lack of structural and mechanistic data. This study investigates the binding and pore formation mechanism of the Plasmodium falciparum PLP1 (PfPLP1), which is expressed during the human life cycle of the parasite. We modeled PfPLP1 structure and performed both all-atom and coarse-grained molecular dynamics simulations in soluble and membrane-associated states. PfPLP1 comprises two domains, a canonical MACPF domain and a {beta}-pleated sheet domain-apicomplexan perforin {beta}-domain (APC{beta}). Initial membrane binding is mediated by cationic residues at the base of the APC{beta} domain, which interact with the polar headgroups of the lipids from the host cell membrane. We analyzed the membrane-inserted tetrameric form where water molecules were observed to penetrate between the tetramer and the lipid bilayer, initiating pore opening. During this process, lipids reorganize into a toroidal edge to shield their hydrophobic tails, while water mixes with lipid headgroups in a disordered, heterogeneous fashion. Larger oligomeric assemblies show lateral displacement of lipids and a clear tendency to form pore-like structures. This study provides molecular insights into PfPLP1s membrane binding and pore-forming behavior in both monomeric and oligomeric forms. The outcome of this study would be applicable in understanding pore formation mechanism in other PLPs and similar toxins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/682298v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@181ad3borg.highwire.dtl.DTLVardef@940471org.highwire.dtl.DTLVardef@1af9c23org.highwire.dtl.DTLVardef@33fd45_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIStructure of Plasmodium falciparum PLP 1 (PfPLP1) as soluble form and membrane-inserted form. C_LIO_LIPfPLP1 initiates pore formation by binding to the host cell membrane with the help of the APC-{beta} domain through cationic residues. C_LIO_LISmaller oligomers of membrane-inserted PfPLP1 form an arc-like structure by reorganizing membrane lipids into a toroidal edge. C_LIO_LIDecamer and higher oligomers of membrane-inserted PfPLP1 form a pore-like structure by lateral displacement of the membrane lipids. C_LI

biochemistry↗

bioRxiv2025

Rewiring Catalytic Craters: A Path for Engineering β-Glucosidases to Improve Glucose Tolerance

Bioethanol, a sustainable alternative to fossil fuels, is produced from cellulose via enzymatic saccharification. {beta}-Glucosidase, a key enzyme in this process, hydrolyses disaccharides into glucose but is limited by feedback inhibition. This study investigated GH1 {beta}-glucosidase from a soil metagenome (UnBGl1) to understand and overcome this limitation. We solved the near-atomic resolution crystal structure of UnBGl1 at 1.15 [A] as an apo enzyme and report the first high-resolution crystal structures of the enzyme in its pre-hydrolytic state as a cellobiose complex and covalent intermediate-bound state, capturing key stages of its catalytic mechanism. Structural analysis revealed three crucial glucose-binding subsites in UnBGl1s crater. Glucose binding at the -1 subsite induced a 1.4 [A] shift in E370, increasing the distance between catalytic glutamates and reducing enzymatic activity. The C188V variant, generated by rational engineering, significantly improved glucose tolerance to 2.5 M. Additionally, the H261W mutation at the +2 subsite enhanced kinetic properties by improving cellobiose affinity (Km = 22.87 {+/-} 1.1 mM) and shifted the optimal pH to 5.5 from 6.0. Comparative structural analysis with other glucose-tolerant GH1 {beta}-glucosidases revealed conserved residues at the -1 subsite crucial for substrate stabilization and +1 subsite residues interacting with glucose, offering targets for further optimization. Engineered UnBGl1 variants retained high stability and activity on sugarcane bagasse, demonstrating their potential for industrial cellulase cocktails. These findings provide a robust framework for engineering {beta}-glucosidases with enhanced glucose tolerance and catalytic efficiency, paving the way for improved bioethanol production and contributing to sustainable energy solutions.

biochemistry↗

bioRxiv2024

Structural insights and rational design of Pseudomonas putida KT2440 Omega transaminases for enhanced biotransformation of (R)-Phenylacetylcarbinol to (1R, 2S)-Norephedrine

Omega transaminases ({omega}-TAs) can mediate the chiral amination of several unnatural substrates without the requirement of an -COOH group, and are highly relevant in the production of several pharmaceutical intermediates of commercial interest. Development of better variants of {omega}-TAs are hence essential for their industrial uses. We have studied the active site architecture of the wild-type {omega}-TAs, to develop engineered enzymes for enhancing the biotransformation of (R)-Phenylacetylcarbinol to (1R, 2S)-Norephedrine. Two such {omega}-TAs (TA_5182 and TA_2799) from P. putida KT2440 strain were overexpressed and purified as recombinant proteins. Crystal structures of TA_5182 were solved in two conformations, and significant movements of two highly flexible loops were observed in these different states. The TA_2799 structure was determined in the co-factor bound state with a PLP molecule covalently bonded to the catalytic K286 as an internal aldimine. Enzyme assays indicated that TA_2799 required significantly higher concentrations of co-factor than TA_5182 to achieve satisfactory biotransformation of (R)-PAC. A key mutation of L322F in TA_2799 drastically reduced the co-factor dependency of the TA_2799_L322F mutant enzyme, and the mutant remained active for 96h at 30{degrees}C. The crystal structure of the mutant enzyme revealed an asparagine residue that mediates a hydrogen bonding network at the dimeric interface of the enzyme and is absent in TA_5182. The TA_5182_G119N mutant also showed enhanced co-factor affinity. The results of our studies will help generate Pseudomonad {omega}-TAs and {omega}-TAs from other organisms with high efficiency for asymmetric synthesis, to be used in host systems for optimal large-scale industrial biotransformation.

biochemistry↗

bioRxiv2024

Faster amylin aggregation on fibrillar collagen hastens diabetic progression through β cell death and loss of function

Amyloid deposition of the neuroendocrine peptide amylin in islet tissues is a hallmark of type 2 diabetes (T2DM), leading to {beta}-cell toxicity through nutrient deprivation, membrane rupture and apoptosis. Though accumulation of toxic amylin aggregates in islet matrices is well documented, the role of the islet extracellular matrix in mediating amylin aggregation and its pathological consequences remains elusive. Here, we address this question by probing amylin interaction with collagen I (Col I)--whose expression in the islet tissue increases during diabetes progression. By combining multiple biophysical techniques, we show that hydrophobic, hydrophilic & cation-{pi} interactions regulate amylin binding to Col I, with fibrillar collagen driving faster amylin aggregation. Amylin-entangled Col I matrices containing high amounts of amylin induce death and loss of function of INS1E {beta}-cells. Together, our results illustrate how amylin incorporation in islet matrices through amylin-Col interactions drives T2DM progression by impacting {beta}-cell viability and insulin secretion.

biophysics↗

bioRxiv2024

Rational design paving the way for improving glucose tolerance and catalytic properties of a β-glucosidase from Acetivibrio thermocellus

Cellulases are an ensemble of enzymes that hydrolyse cellulose chains to fermentable glucose, hence, are widely used in bioethanol production. The last enzyme of the cellulose degradation pathway - {beta}-glucosidase, is inhibited by its product - glucose. The product inhibition by glucose hinders cellulose hydrolysis limiting the saccharification during bioethanol production. Therefore, engineered {beta}-glucosidases with improved glucose tolerance along with the catalytic efficiency are the need of the hour. This study focuses on the rational engineering of {beta}-glucosidase from Acetivibrio thermocellus (WT-AtGH1). Recombinant WT-AtGH1 exhibited activity on cellobiose and p-nitrophenyl-{beta}-D-glucosidase as substrates and retained around 80% of its activity over 48 hours at 55{degrees}C, pH 5.5. However, WT-AtGH1 showed low glucose tolerance of 380 mM as compared to the required IC50 value of > 800 mM for industrial use. Therefore, the rational design approach was applied for improving the glucose tolerance of this enzyme. We determined 3 [A] resolution crystal structure of WT-AtGH1. The structure-based engineered G168W-AtGH1 and S242W-AtGH1 mutants exhibited improved glucose tolerance of 840 mM and 612 mM, respectively. Surprisingly, S242L-AtGH1 mutant showed [~] 2.5-fold increase in the catalytic efficiency as compared to WT-AtGH1. A combinatorial effect of improved glucose tolerance, as well as enhanced catalytic efficiency, was observed for the G168W-S242L-AtGH1 mutant. All the mutants with enhanced properties showed considerable stability at industrial operating conditions of 55{degrees}C and pH 5.5. Thus, we present the next-generation mutants of WT-AtGH1 with improved glucose tolerance and kinetic properties that have the potential to increase the efficiency of the saccharification process for second generation bioethanol production.

biochemistry↗

bioRxiv2024

Molecular insights into the oligosaccharide binding, recognition and hydrolysis by a fungal exo-beta-(1,3)-glucanase

Carbohydrate hydrolysing enzymes assume special industrial and commercial interest as a source for yielding fermentable glucose especially for the biofuel industry. Among these enzymes, the exo-{beta}-(1,3) glucanases are promising for industrial use as they hydrolyze sugars such as laminarin, a major constituent of the algal cell wall. Exploring the structure and function of these enzymes is of particular interest for the improvement of their functional properties for industrial use. We report the structural and biochemical characterizations of Aspergillus oryzae exo-{beta}-(1,3) glucanase (AoBgl). We have expressed, purified, and performed biochemical characterizations of the recombinant AoBgl. Purified AoBgl is found to hydrolyse {beta}-(1,3)-glycosidic linkages present in the oligosaccharide laminaritriose and the polysaccharide, laminarin effectively while retaining >50% activity at glucose concentrations of around 1.5M. We have determined three high-resolution structures of AoBgl: (a) apo form at 1.75 [A], (b) complexed form with bound disaccharide at 1.73 [A] and (c) glucose-bound form at 1.20 [A]. Sequence analysis and structural comparison indicate that AoBgl belongs to the GH5 sugar hydrolase family. The sugar-bound structures reveal the mode of substrate binding and interactions at the active site of AoBgl. Further, molecular dynamics simulation and mutational studies indicate that AoBgl can effectively bind trisaccharides and higher oligosaccharides. Our biochemical and structural data provide detailed molecular insights into the active site of this GH5 enzyme and would be helpful in the rational engineering of glycosyl hydrolases belonging to similar families for industrial use.

biochemistry↗

bioRxiv2024

Conformational dynamics associated with remote residues regulate the kinetic properties of homologous glutamate dehydrogenases (GDHs)

Glutamate dehydrogenase (GDH) is a key enzyme in all living organisms and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. The crystal structures and first-ever cryo-EM structures of apo-AtGDH captured arrays of conformational ensembles. Comparative structural analysis has revealed a wider mouth opening in allosteric AnGDH ([~] 21 [A]) compared to non-allosteric AtGDH ([~]17 [A]) in their apo states. A network of interaction related to the amino acid substitutions in Domain II is responsible for differential structural dynamics in these GDHs. Remarkably, we identified one remotely located substitution in Domain II, i.e., R246 to S, a part of the network, which reversed the kinetic properties of AtGDH into an allosteric one and controls the mouth opening. Our data also indicate that dynamic discrepancy influences the substrate binding affinity and catalytic activity in AnGDH and AtGDH. We have successfully demonstrated for the first time, that remotely located residues and the conformational dynamics regulate the kinetic properties in homologous GDHs.

biochemistry↗

bioRxiv2023

Toxoplasma gondii aspartic protease 5 (TgASP5): Understanding structural details and inhibition mechanism.

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 (TgASP5) 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 TgASP5 mature enzyme. Molecular modeling and all-atom simulation provided in-depth knowledge of the active site architecture of TgASP5. 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 RRLStatine. Screening the known aspartic protease inhibitors against TgASP5 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. TgASP5 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 TgASP5.

bioinformatics↗

bioRxiv2021

Inter-subunit Crosstalk via PDZ Synergistically Governs Allosteric Activation of Proapoptotic HtrA2

Mitochondrial serine protease - High temperature requirement A2 (HtrA2), is associated with various diseases including neurodegenerative disorders and cancer. Despite availability of structural details, the reports on HtrA2s mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound the role of regulatory PDZ domains in promoting synergistic coordination between HtrA2 subunits, we generated heterotrimeric HtrA2 variants comprising different numbers of PDZs and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual activity in a way that proffered a hypothesis advocating intermolecular allosteric crosstalk via PDZ domains in trimeric HtrA2. Furthermore, structural and computational snapshots affirmed the role of PDZs in secondary structural element formation and coordinated reorganization of the N-terminal region and regulatory loops. Therefore, apart from providing cues for devising structure-guided therapeutic strategies, this study establishes a physiologically relevant working model of complex allosteric regulation through a multifaceted trans-mediated cooperatively-shared energy landscape.

biochemistry↗

bioRxiv2021

Molecular insights into the inhibition of glutamate dehydrogenase (GDH) by the dicarboxylic acid metabolites

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+-dependent reversible conversion of -ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+-dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

biophysics↗

bioRxiv2021

Molecular insights into the inhibition of plasmepsins by HIV-1 protease inhibitors: Implications for antimalarial drug discovery

1.Malaria is a deadly disease, and the worldwide emergence of drug resistance in the Plasmodium parasites demands the development of novel and potent antimalarials. HIV-1 protease inhibitors (HIV-1 PIs) alleviate the Plasmodium pathogenesis during malaria/HIV-1 co-infection plausibly by inhibiting vacuolar plasmepsins (PMs), the hemoglobin degrading proteases from P. falciparum. All five FDA-approved HIV-1 PIs tested against PMII exhibit the Ki values in the low micromolar range of which RTV and LPV display the highest inhibition activity. Both inhibitors impede in vitro growth of P. falciparum at low micromolar IC50 values. We report the first crystal structures of PMII complexed with RTV and LPV that reveal the binding mode and interactions of the inhibitors in the active site as well as elucidate the mechanism underlying their differential potency. The conformational flexibility of the P4 group in RTV allows it to explore multiple regions of the S4 pocket. The present study establishes vacuolar PMs as potential drug targets of HIV-1 PIs. The molecular details explaining the inhibitory mechanism of HIV-1 PIs might be crucial in designing novel and potent antimalarial analogs. This work strengthens the prospect of drug repurposing as an alternative strategy towards antimalarial treatments and provides an opportunity to tackle malaria and HIV-1 co-infection.

biochemistry↗

bioRxiv2021

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

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 are 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.

biophysics↗

bioRxiv2020

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-{pi} interaction, governing formation of the transmembrane {beta}-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with loss of pore forming ability of Ply facilitating a benign association of SPN in an alternative, intracellular host niche. AUTHOR SUMMARYStreptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by expressing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

microbiology↗