Molecular insights into the inhibition of glutamate dehydrogenase by the dicarboxylic acid metabolites.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+ -dependent reversible conversion of α-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.

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-π interaction, governing formation of the transmembrane ß-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 Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Structural basis for the catalytic mechanism and α-ketoglutarate cooperativity of glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior. We determined the first high-resolution crystal structures of glutamate dehydrogenase from the fungus Aspergillus niger (AnGDH), a unique NADP+-dependent allosteric enzyme that is forward-inhibited by the formation of mixed disulfide. We determined the structures of the active enzyme in its apo form and in binary/ternary complexes with bound substrate (α-ketoglutarate), inhibitor (isophthalate), coenzyme (NADPH), or two reaction intermediates (α-iminoglutarate and 2-amino-2-hydroxyglutarate). The structure of the forward-inhibited enzyme (fiAnGDH) was also determined. The hexameric AnGDH had three open subunits at one side and three partially closed protomers at the other, a configuration not previously reported. The AnGDH hexamers having subunits with different conformations indicated that its α-ketoglutarate-dependent homotropic cooperativity follows the Monod-Wyman-Changeux (MWC) model. Moreover, the position of the water attached to Asp-154 and Gly-153 defined the previously unresolved ammonium ion-binding pocket, and the binding site for the 2'-phosphate group of the coenzyme was also better defined by our structural data. Additional structural and mutagenesis experiments identified the residues essential for coenzyme recognition. This study reveals the structural features responsible for positioning α-ketoglutarate, NADPH, ammonium ion, and the reaction intermediates in the GDH active site.

Structural modulation of a periplasmic sugar-binding protein probes into its evolutionary ancestry.

Substrate-binding proteins (SBPs) are periplasmic proteins consisting of two α/ß domains joined by a hinge region with specificity towards cognate ligands. Based on three-dimensional fold, sugar-specific SBPs have been classified into cluster B and cluster D-I. The analysis of sequences and structures of sugar-binding pocket of cluster D-I SBPs revealed the presence of extra residues on two loops (L1, L2) and a helix (H1) in few members of this family, that binds specifically to monosaccharides. Presence of conserved histidine in L2 and tryptophan in H1 can be considered as the identity marks for the cluster D-I monosaccharide-binding SBPs. A glucose binding protein (ppGBP) from Pseudomonas putida CSV86 was found to contain a structural fold similar to oligosaccharide-binding cluster D-I SBPs, but functionally binds to only glucose due to constriction of its binding pocket mainly by L2 (375-382). ppGBP with partial deletion of L2 (ppGBPΔL2) was created, crystallized and biochemical characterization was performed. Compared to wild type ppGBP, the ppGBPΔL2 structure showed widening of the glucose-binding pocket with ∼80% lower glucose binding. Our results show that the substrate specificity of SBPs can be altered by modulating the size of the binding pocket. Based on this, we propose a sub classification of cluster D-I SBPs into (i) cluster D-I(a)-monosaccharide-binding SBPs and (ii) cluster D-I(b)-oligosaccharide-binding SBPs. This study also provides the direct structural and functional correlation indicating that divergence of proteins may occur through insertions or deletions of sequences in the already existing SBPs leading to evolution at the functional level.

High Resolution Structures of Periplasmic Glucose-binding Protein of Pseudomonas putida CSV86 Reveal Structural Basis of Its Substrate Specificity.

Periplasmic substrate-binding proteins (SBPs) bind to the specific ligand with high affinity and mediate their transport into the cytoplasm via the cognate inner membrane ATP-binding cassette proteins. Because of low sequence identities, understanding the structural basis of substrate recognition by SBPs has remained very challenging. There are several structures available for the ligand-bound sugar SBPs, but very few unliganded structures are reported. No structural data are available for sugar SBPs fromPseudomonassp. to date. This study reports the first high resolution crystal structures of periplasmic glucose-binding protein fromPseudomonas putidaCSV86 (ppGBP) in unliganded form (2.5 Å) and complexed with glucose (1.25 Å) and galactose (1.8 Å). Asymmetric domain closure of ppGBP was observed upon substrate binding. The ppGBP was found to have an affinity of ∼ 0.3 µmfor glucose. The structural analysis showed that the sugars are bound to the protein mainly by hydrogen bonds, and the loss of two strong hydrogen bonds between ppGBP and galactose compared with glucose may be responsible for lowering its affinity toward galactose. The higher stability of ppGBP-glucose complex was also indicated by an 8 °C increase in the melting temperature compared with unliganded form and ppGBP-galactose complex. ppGBP binds to monosaccharide, but the structural features revealed it to have an oligosaccharide-binding protein fold, indicating that during evolution the sugar binding pocket may have undergone structural modulation to accommodate monosaccharide only.

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II.

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.

Elucidating the role of disulfide bond on amyloid formation and fibril reversibility of somatostatin-14: relevance to its storage and secretion.

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys(3)-Cys(14)) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.

Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300.

Members of the C/EBP family of transcription factors bind to the Taz2 domain of p300/CBP and mediate its phosphorylation through the recruitment of specific kinases. Short sequence motifs termed homology boxes A and B, which comprise their minimal transactivation domains (TADs), are conserved between C/EBP activators and are necessary for specific p300/CBP binding. A possible mode of interaction between C/EBP TADs and the p300 Taz2 domain was implied by the crystal structure of a chimeric protein composed of residues 1723-1818 of p300 Taz2 and residues 37-61 of C/EBPℇ. The segment corresponding to the C/EBPℇ TAD forms two orthogonally disposed helices connected by a short linker and interacts with the core structure of Taz2 from a symmetry-related molecule. It is proposed that other members of the C/EBP family interact with the Taz2 domain in the same manner. The position of the C/EBPℇ peptide on the Taz2 protein interaction surface suggests that the N-termini of C/EBP proteins are unbound in the C/EBP-p300 Taz2 complex. This observation is in agreement with the known location of the docking site of protein kinase HIPK2 in the C/EBPß N-terminus, which associates with the C/EBPß-p300 complex.

Toxoplasma gondii aspartic protease 5: structural basis of substrate binding and inhibition mechanism.

Toxoplasma gondii, a worldwide prevalent parasite is responsible for causing toxoplasmosis in almost all warm-blooded animals, including humans. Golgi-resident T. gondii aspartic protease 5 (TgASP5) plays an essential role in the maturation and export of the effector proteins those modulate the host immune system to establish a successful infection. Hence, inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. This is the first report of the detailed structural investigations of the TgASP5 mature enzyme using molecular modeling and an all-atom simulation approach which provide in-depth knowledge of the active site architecture of TgASP5. The analysis of the binding mode of the TEXEL (Toxoplasma EXport Element) substrate to TgASP5 highlighted the importance of the active site residues. Ser505, Ala776 and Tyr689 in the S2 binding pocket are responsible for the specificity towards Arg at the P2 position of TEXEL substrate. The molecular basis of inhibition by the only known inhibitor RRLStatine has been identified, and our results show that it blocks the active site by forming a hydrogen bond with a catalytic aspartate. Besides that, known aspartic protease inhibitors were screened against TgASP5 using docking, MD simulations and MM-PBSA binding energy calculations. The top-ranked inhibitors (SC6, ZY1, QBH) showed higher binding energy than RRLStatine. Understanding the structural basis of substrate recognition and the binding mode of these inhibitors will help to develop potent mechanistic inhibitors against TgASP5. This study will also provide insights into the structural features of pepsin-like aspartic proteases from other apicomplexan parasites for developing antiparasitic agents.Communicated by Ramaswamy H. Sarma.

Inhibition of Plasmodium falciparum plasmepsins by drugs targeting HIV-1 protease: A way forward for antimalarial drug discovery.

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.

Structural studies of vacuolar plasmepsins.

Plasmepsins (PMs) are pepsin-like aspartic proteases present in different species of parasite Plasmodium. Four Plasmodium spp. (P. vivax, P. ovale, P. malariae, and the most lethal P. falciparum) are mainly responsible for causing human malaria that affects millions worldwide. Due to the complexity and rate of parasite mutation coupled with regional variations, and the emergence of P. falciparum strains which are resistant to antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is constant pressure to find new and lasting chemotherapeutic drug therapies. Since many proteases represent therapeutic targets and PMs have been shown to play an important role in the survival of parasite, these enzymes have recently been identified as promising targets for the development of novel antimalarial drugs. The genome of P. falciparum encodes 10 PMs (PMI, PMII, PMIV-X and histo-aspartic protease (HAP)), 4 of which (PMI, PMII, PMIV and HAP) reside within the food vacuole, are directly involved in degradation of human hemoglobin, and share 50-79% amino acid sequence identity. This review focuses on structural studies of only these four enzymes, including their orthologs in other Plasmodium spp.. Almost all original crystallographic studies were performed with PMII, but more recent work on PMIV, PMI, and HAP resulted in a more complete picture of the structure-function relationship of vacuolar PMs. Many structures of inhibitor complexes of vacuolar plasmepsins, as well as their zymogens, have been reported in the last 15 years. Information gained by such studies will be helpful for the development of better inhibitors that could become a new class of potent antimalarial drugs. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Structure and mechanism of the saposin-like domain of a plant aspartic protease.

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

Inter-subunit crosstalk via PDZ synergistically governs allosteric activation of proapoptotic HtrA2.

The 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 HtrA2's mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound the role of regulatory PDZ (Postsynaptic density-95/Discs large/Zonula occludens-1) domains in multimodal activation of HtrA2, 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 inter-molecular allosteric crosstalk via PDZs in HtrA2. Furthermore, structural and computational snapshots affirmed the role of PDZs in secondary structural element formation around the regulatory loops and coordinated reorganization of the N-terminal region. 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 trans-mediated cooperatively shared energy landscape.

Structures of plasmepsin X from Plasmodium 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 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.

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum.

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 Å resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum.

Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite Plasmodium falciparum, and the only one with no crystal structure reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.4 and 3.1Å, respectively. The apoenzyme crystallized in the orthorhombic space group P2(1)2(1)2(1) with two molecules in the asymmetric unit and the structure has been refined to the final R-factor of 20.7%. The KNI-10006 bound enzyme crystallized in the tetragonal space group P4(3) with four molecules in the asymmetric unit and the structure has been refined to the final R-factor of 21.1%. In the PMI-KNI-10006 complex, the inhibitors were bound identically to all four enzyme molecules, with the opposite directionality of the main chain of KNI-10006 relative to the direction of the enzyme substrates. Such a mode of binding of inhibitors containing an allophenylnorstatine-dimethylthioproline insert in the P1-P1' positions, previously reported in a complex with PMIV, demonstrates the importance of satisfying the requirements for the proper positioning of the functional groups in the mechanism-based inhibitors towards the catalytic machinery of aspartic proteases, as opposed to binding driven solely by the specificity of the individual enzymes. A comparison of the structure of the PMI-KNI-10006 complex with the structures of other vacuolar plasmepsins identified the important differences between them and may help in the design of specific inhibitors targeting the individual enzymes.

Advancements in macromolecular crystallography: from past to present.

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.

Activation mechanism of plasmepsins, pepsin-like aspartic proteases from Plasmodium, follows a unique trans-activation pathway.

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.

Cytotoxicity of apo bovine α-lactalbumin complexed with La3+ on cancer cells supported by its high resolution crystal structure.

Cancer remains one of the biggest threats to human society. There are massive demands for compounds to selectively kill cancerous cells. Earlier studies have shown that bovine α -lactalbumin made lethal to tumor cells (BAMLET) becomes cytotoxic against cancer cells in complex with oleic acid {Hoque, M. et. al., PLoSOne 8, e68390 (2013)}. In our study, we obtained bovine α-lactalbumin complexed with lanthanum ion (La3+-B-α-LA) and determined its high resolution crystal structure. The natural calcium binding site of bovine α-lactalbumin is replaced by lanthanum. The La3+ complex formation by B-α-apo-LA was also supported by various biophysical methods. Interestingly, our complex, La3+-B-α-LA exhibits much greater anticancer activity against breast cancer cells as compared to the reported BAMLET-oleic acid complex. This study shows that La3+-B-α-LA complex is preferentially more toxic to MCF-7 cells as compared to KB (oral cancer) and HeLa (cervical) cells, while almost non-toxic to the healthy cells that we studied. Our data indicates that the cytotoxicity of La3+-B-α-LA against cancer cells is through apoptotic path way. The higher anticancer activity of La3+-B-α-LA is attributable to the requisite structural changes induced in the protein by La3+ binding as supported by the crystal structure of the complex.

The catalysis of the 1,1-proton transfer by alpha-methyl-acyl-CoA racemase is coupled to a movement of the fatty acyl moiety over a hydrophobic, methionine-rich surface.

Alpha-methylacyl-CoA racemases are essential enzymes for branched-chain fatty acid metabolism. Their reaction mechanism and the structural basis of their wide substrate specificity are poorly understood. High-resolution crystal structures of Mycobacterium tuberculosis alpha-methylacyl-CoA racemase (MCR) complexed with substrate molecules show the active site geometry required for catalysis of the interconversion of (2S) and (2R)-methylacyl-CoA. The thioester oxygen atom and the 2-methyl group are in a cis-conformation with respect to each other. The thioester oxygen atom fits into an oxyanion hole and the 2-methyl group points into a hydrophobic pocket. The active site geometry agrees with a 1,1-proton transfer mechanism in which the acid/base-pair residues are His126 and Asp156. The structures of the complexes indicate that the acyl chains of the S-substrate and the R-substrate bind in an S-pocket and an R-pocket, respectively. A unique feature of MCR is a large number of methionine residues in the acyl binding region, located between the S-pocket and the R-pocket. It appears that the (S) to (R) interconversion of the 2-methylacyl chiral center is coupled to a movement of the acyl group over this hydrophobic, methionine-rich surface, when moving from its S-pocket to its R-pocket, whereas the 2-methyl moiety and the CoA group remain fixed in their respective pockets.