Allostery Wiring Map for Kinesin Energy Transduction and its Evolution [Enzymology]

August 8th, 2016 by Richard, J., Kim, E. D., Nguyen, H., Kim, C. D., Kim, S.

How signals between the kinesin active- and cytoskeletal-binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and, thus, are predicted to form a complex, nonlinear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop-5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily-derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein, rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases [DNA and Chromosomes]

August 8th, 2016 by Vashishtha, A. K., Wang, J., Konigsberg, W. H.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3' hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group making it a highly effective nucleophile that can attack the alpha phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly two metal ions are required for proof-reading, one helps to lower the pKa of the attacking water molecule and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all of the effects of these different metal ions can be rationalized based on their intrinsic properties which are tabulated in this review.

Rice endosperm starch phosphorylase (Pho1) assembles with disproportionating enzyme (Dpe1) to form a protein complex that enhances synthesis of malto-oligosaccharides [Metabolism]

August 8th, 2016 by Hwang, S.-K., Koper, K., Satoh, H., Okita, T. W.

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pull-down experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multi-enzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger malto-oligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30oC. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.
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Functional Annotation of a Presumed Nitronate Monooxygenase Reveals a New Class of NADH:quinone Reductases [Enzymology]

August 8th, 2016 by Ball, J., Salvi, F., Gadda, G.

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi-Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half reaction with NADH showed a kred value of 24 s-1 and an apparent Kd value estimated in the low μM range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase.

An oral load of [13C3]glycerol and blood NMR analysis detect fatty acid esterification, pentose phosphate pathway and glycerol metabolism through the tricarboxylic acid cycle in human liver [Lipids]

July 18th, 2016 by Jin, E. S., Sherry, A. D., Malloy, C. R.

Drugs and other interventions for high-impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-13C3]glycerol and blood was dawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution 13C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-13C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state, but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis and flux through anaplerotic pathways in mitochondria of human liver.
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High affinity binding of the receptor-associated protein D1D2 domains with the LDL receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysine 60 and 191. [Molecular Biophysics]

July 11th, 2016 by Prasad, J. M., Young, P. A., Strickland, D. K.

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three domain protein that contains two high affinity binding sites for LRP1: one located within domains 1 and 2, and one located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2/LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor.
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NEDD4L Catalyzes Ubiquitination of PIK3CA and Regulates PI3K-AKT Signaling [Signal Transduction]

June 23rd, 2016 by Wang, Z., Dang, T., Liu, T., Chen, S., Li, L., Huang, S., Fang, M.

Oncogenic PIK3CA (p110α), the catalytic subunit of class IA PI3K, plays a major role in PI3K related cancer progression. The mechanisms underlying the dynamic regulation of PIK3CA protein levels remain unknown. Here we demonstrated that PIK3CA is regulated by polyubiquitination. We identified NEDD4L as the E3 ligase that catalyzes PIK3CA polyubiquitination, leading to its proteasome-dependent degradation. NEDD4L ubiquitinates both the free and regulatory subunit-bound PIK3CA, but does not ubiquitinate the regulatory subunit of PI3K. Overexpression of NEDD4L accelerates the turnover rate of PIK3CA whereas suppression of NEDD4L results in not only the accumulation of PIK3CA but also a paradoxical decrease of AKT activation. Thus, we propose that NEDD4L negatively regulates PIK3CA protein levels via ubiquitination and is required for the maintenance of PI3K-AKT signaling pathway.

Sialylation of GPI Anchors of Mammalian Prions is Regulated in a Host-, Tissue- and Cell-Specific Manner [Glycobiology and Extracellular Matrices]

June 17th, 2016 by Katorcha, E., Srivastava, S., Klimova, N., Baskakov, I. V.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrPC. PrPC is posttranslationally modified with N-linked glycans and a sialylated glycosylinositol phospholipid anchor (GPI). Conformational conversion of PrPC give rise to glycosylated and GPI-anchored PrPSc. The question of the sialylation status of GPIs within PrPSc has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrPSc with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrPC with sialylo-GPIs could be recruited into PrPSc, whereas PrPC with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed sialylation status of GPIs within PrPSc generated in brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrPC with both sialo- and asialo-GPIs is a common feature of PrPSc. The mixtures of sialo- and asialo-GPIs were observed in PrPSc universally regardless of prion strain, as well as host, tissue or type of cells that produced PrPSc. Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue and cell type, but not prion strain. In summary, the current study found no strain-specific preferences for selecting PrPC with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrPSc is regulated in a cell-, tissue- or host-specific manner, and is likely to be determined by the specifics of GPI biosynthesis.
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Dual Function of Phosphoubiquitin in Activation of Parkin [Molecular Bases of Disease]

June 9th, 2016 by Walinda, E., Morimoto, D., Sugase, K., Shirakawa, M.

Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C-terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis. Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL- binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2~Ub binding triggers large-scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin/E2~Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin.

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: Insights on Leucine and Aliphatic Glucosinolate Biosynthesis [Protein Structure and Folding]

May 2nd, 2016 by Lee, S. G., Nwumeh, R., Jez, J. M.

Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different beta-hydroxyacids in the leucine and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg2+ or NAD+. These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the beta-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.
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