Insights into the Substrate Specificity of a Thioesterase Rv0098 of Mycobacterium Tuberculosis through X-ray Crystallographic and Molecular Dynamics Studies

The crystal structure of Rv0098, a long-chain fatty acyl-CoA thioesterase from Mycobacterium tuberculosis with bound dodecanoic acid at the active site provided insights into the mode of substrate binding but did not reveal the structural basis of substrate specificities of varying chain length. Molecular dynamics studies demonstrated that certain residues of the substrate binding tunnel are flexible and thus modulate the length of the tunnel. The flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. A combination of crystallographic and molecular dynamics studies thus explained the structural basis of accommodating long chain substrates by Rv0098 of M. tuberculosis.

Mutations in hpyAVIBM, C5 cytosine DNA methyltransferase from Helicobacter pylori result in relaxed specificity

The genome of Helicobacter pylori is rich in restrictionmodification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C5 cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nucleotide sequence result in proteins with new properties.

Structural and Mutational Studies on Substrate Specificity and Catalysis of Salmonella typhimurium D-Cysteine Desulfhydrase

Salmonella typhimurium DCyD (StDCyD) is a fold type II pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. It also efficiently degrades beta-chloro-D-alanine (beta CDA). D-Ser is a poor substrate while the enzyme is inactive with respect to L-Ser and 1-amino-1-carboxy cyclopropane (ACC). Here, we report the X-ray crystal structures of StDCyD and of crystals obtained in the presence of D-Cys, beta CDA, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS) at resolutions ranging from 1.7 to 2.6 angstrom. The polypeptide fold of StDCyD consisting of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) resembles other fold type II PLP dependent enzymes. The structures obtained in the presence of D-Cys and beta CDA show the product, pyruvate, bound at a site 4.0-6.0 angstrom away from the active site. ACC forms an external aldimine complex while D- and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at C alpha proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggest formation of PMP by the hydrolysis of cycloserines. Mutational studies suggest that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for C alpha proton abstraction from D-Cys. Based on these studies, a probable mechanism for the degradation of D-Cys by StDCyD is proposed.

IDH1 Mutations in Diffusely Infiltrating Astrocytomas Grade Specificity, Association With Protein Expression, and Clinical Relevance

IDH1 mutations are frequent genetic alterations in low-grade diffuse gliomas and secondary glioblastoma (GBM). To validate mutation frequency, IDH1 gene at codon 132 was sequenced in 74 diffusely infiltrating astrocytomas: diffuse astrocytoma (DA; World Health Organization WHO] grade II), anaplastic astrocytoma (AA; WHO grade III), and GBM (WHO grade IV). All cases were immunostained with IDH1-R132H monoclonal antibody. Mutational status was correlated with mutant protein expression, patient age, duration of symptoms, and prognosis of patients with GBM. We detected 31 (41.9%) heterozygous IDH1 mutations resulting in arginine-to-histidine substitution (R132H;CGT-CAT). All 12 DAs (100%), 13 of 14 AAs (92.9%), and 6 of 48 GBMs (12.5%) (5/6 83.3%] secondary, and 1/42 2.4%] primary) harbored IDH1 mutations. The correlation between mutational status and protein expression was significant (P < .001). IDH1 mutation status, though not associated with prognosis of patients with GBM, showed significant association with younger age and longer duration of symptoms in the whole cohort (P < .001). Our study validates IDH1 mutant protein expression across various grades of astrocytoma, and demonstrates a high incidence of IDH1 mutations in DA, AA, and secondary GBM.

The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate Methanol Metabolism antagonistically in pichia pastoris

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5' CYCCNY 3' motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species.

Ca2+ Binding to the ExDxD Motif Regulates the DNA Cleavage Specificity of a Promiscuous Endonuclease

Most of the restriction endonucleases (REases) are dependent on Mg2+ for DNA cleavage, and in general, Ca2+ inhibits their activity. RKpnI, an HNH active site containing beta beta alpha-Me finger nuclease, is an exception. In presence of Ca2+, the enzyme exhibits high-fidelity DNA cleavage and complete suppression of Mg2+-induced promiscuous activity. To elucidate the mechanism of unusual Ca2+-mediated activity, we generated alanine variants in the putative Ca-2+ binding motif, E(132)xD(134)xD(136), of the enzyme. Mutants showed decreased levels of DNA cleavage in the presence of Ca2+. We demonstrate that ExDxD residues are involved in Ca2+ coordination; however, the invariant His of the catalytic HNH motif acts as a general base for nucleophile activation, and the other two active site residues, D148 and Q175, also participate in Ca2+-mediated cleavage. Insertion of a 10-amino acid linker to disrupt the spatial organization of the ExDxD and HNH motifs impairs Ca2+ binding and affects DNA cleavage by the enzyme. Although ExDxD mutant enzymes retained efficient cleavage at the canonical sites in the presence of Mg2+, the promiscuous activity was greatly reduced, indicating that the carboxyl residues of the acidic triad play an important role in sequence recognition by the enzyme. Thus, the distinct Ca2+ binding motif that confers site specific cleavage upon Ca2+ binding is also critical for the promiscuous activity of the Mg2+-bound enzyme, revealing its role in metal ion-mediated modulation of DNA cleavage.

Generation of Ligand Specificity and Modes of Oligomerization in beta-Prism I Fold Lectins

beta-Prism I fold lectins constitute one of the five widely occurring structural classes of plant lectins. Each single domain subunit is made up of three Greek key motifs arranged in a threefold symmetric fashion. The threefold symmetry is not reflected in the sequence except in the case of the lectin from banana, a monocot, which carries two sugar-binding sites instead of the one in other lectins of known three-dimensional structure, all from dicots. This is believed to be a consequence of the different evolutionary paths followed by the lectin in monocots and dicots. The galactose-specific lectins among them have two chains produced by posttranslational proteolysis and contain three aromatic residues at the binding site. The extended binding sites of galactose- and mannose-specific lectins have been thoroughly characterized. Ligand binding at the sites involves both conformational selection and induced fit. Molecular plasticity of some of the lectins in the family has been characterized. The plasticity appears to be such as to promote variability in quaternary association which could be dimeric, tetrameric, or octameric. Structural and evolutionary reasons for the variability have been explored, and the relation of oligomerization to ligand binding and conformational selection investigated.

Mechanism design for time critical and cost critical task execution via crowdsourcing

An exciting application of crowdsourcing is to use social networks in complex task execution. In this paper, we address the problem of a planner who needs to incentivize agents within a network in order to seek their help in executing an atomic task as well as in recruiting other agents to execute the task. We study this mechanism design problem under two natural resource optimization settings: (1) cost critical tasks, where the planner's goal is to minimize the total cost, and (2) time critical tasks, where the goal is to minimize the total time elapsed before the task is executed. We identify a set of desirable properties that should ideally be satisfied by a crowdsourcing mechanism. In particular, sybil-proofness and collapse-proofness are two complementary properties in our desiderata. We prove that no mechanism can satisfy all the desirable properties simultaneously. This leads us naturally to explore approximate versions of the critical properties. We focus our attention on approximate sybil-proofness and our exploration leads to a parametrized family of payment mechanisms which satisfy collapse-proofness. We characterize the approximate versions of the desirable properties in cost critical and time critical domain.

Multi-task learning using shared and task specific information

Multi-task learning solves multiple related learning problems simultaneously by sharing some common structure for improved generalization performance of each task. We propose a novel approach to multi-task learning which captures task similarity through a shared basis vector set. The variability across tasks is captured through task specific basis vector set. We use sparse support vector machine (SVM) algorithm to select the basis vector sets for the tasks. The approach results in a sparse model where the prediction is done using very few examples. The effectiveness of our approach is demonstrated through experiments on synthetic and real multi-task datasets.

Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 mu M mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Functional specificity among yeast mitochondrial Hsp70s paralogs and orthologs

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can “make or break” mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Reach task-associated excitatory overdrive of motor cortical neurons following infusion with ALS-CSF

Converging evidence from transgenic animal models of amyotrophic lateral sclerosis (ALS) and human studies suggest alterations in excitability of the motor neurons in ALS. Specifically, in studies on human subjects with ALS the motor cortex was reported to be hyperexcitable. The present study was designed to test the hypothesis that infusion of cerebrospinal fluid from patients with sporadic ALS (ALS-CSF) into the rat brain ventricle can induce hyperexcitability and structural changes in the motor cortex leading to motor dysfunction. A robust model of sporadic ALS was developed experimentally by infusing ALS-CSF into the rat ventricle. The effects of ALS-CSF at the single neuron level were examined by recording extracellular single unit activity from the motor cortex while rats were performing a reach to grasp task. We observed an increase in the firing rate of the neurons of the motor cortex in rats infused with ALS-CSF compared to control groups. This was associated with impairment in a specific component of reach with alterations in the morphological characteristics of the motor cortex. It is likely that the increased cortical excitability observed in the present study could be the result of changes in the intrinsic properties of motor cortical neurons, a dysfunctional inhibitory mechanism and/or an underlying structural change culminating in a behavioral deficit.

Cooperative Communication of UAV to Perform Multi-task Using Nature Inspired Techniques

In this paper, the approach for assigning cooperative communication of Uninhabited Aerial Vehicles (UAV) to perform multiple tasks on multiple targets is posed as a combinatorial optimization problem. The multiple task such as classification, attack and verification of target using UAV is employed using nature inspired techniques such as Artificial Immune System (AIS), Particle Swarm Optimization (PSO) and Virtual Bee Algorithm (VBA). The nature inspired techniques have an advantage over classical combinatorial optimization methods like prohibitive computational complexity to solve this NP-hard problem. Using the algorithms we find the best sequence in which to attack and destroy the targets while minimizing the total distance traveled or the maximum distance traveled by an UAV. The performance analysis of the UAV to classify, attack and verify the target is evaluated using AIS, PSO and VBA.

Specific Sequence of a Beta Turn in Human La Protein May Contribute to Species Specificity of Hepatitis C Virus

Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a beta-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting beta-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La beta-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein.