scholarly journals Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme

2017 ◽  
Vol 114 (23) ◽  
pp. 6034-6039 ◽  
Author(s):  
Jan Philip Wurm ◽  
Iris Holdermann ◽  
Jan H. Overbeck ◽  
Philipp H. O. Mayer ◽  
Remco Sprangers
Keyword(s):  
Molecular Level ◽  
Structural Information ◽  
Active State ◽  
Transverse Relaxation ◽  
Activator Proteins ◽  
Regulatory Domains ◽  
Conformational Equilibria ◽  
Catalytically Active ◽  
Decapping Enzyme ◽  
Dynamic Equilibria

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5′ cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA.

scholarly journals Diversity and evolution of B-family DNA polymerases

2020 ◽  
Vol 48 (18) ◽  
pp. 10142-10156 ◽  
Author(s):  
Darius Kazlauskas ◽  
Mart Krupovic ◽  
Julien Guglielmini ◽  
Patrick Forterre ◽  
Česlovas Venclovas
Keyword(s):  
Structural Information ◽  
Dna Polymerases ◽  
Comprehensive Analysis ◽  
Metagenomic Data ◽  
Dna Viruses ◽  
Taxonomic Distribution ◽  
Binding Domains ◽  
Catalytically Active ◽  
Domains Of Life ◽  
Massive Accumulation

Abstract B-family DNA polymerases (PolBs) represent the most common replicases. PolB enzymes that require RNA (or DNA) primed templates for DNA synthesis are found in all domains of life and many DNA viruses. Despite extensive research on PolBs, their origins and evolution remain enigmatic. Massive accumulation of new genomic and metagenomic data from diverse habitats as well as availability of new structural information prompted us to conduct a comprehensive analysis of the PolB sequences, structures, domain organizations, taxonomic distribution and co-occurrence in genomes. Based on phylogenetic analysis, we identified a new, widespread group of bacterial PolBs that are more closely related to the catalytically active N-terminal half of the eukaryotic PolEpsilon (PolEpsilonN) than to Escherichia coli Pol II. In Archaea, we characterized six new groups of PolBs. Two of them show close relationships with eukaryotic PolBs, the first one with PolEpsilonN, and the second one with PolAlpha, PolDelta and PolZeta. In addition, structure comparisons suggested common origin of the catalytically inactive C-terminal half of PolEpsilon (PolEpsilonC) and PolAlpha. Finally, in certain archaeal PolBs we discovered C-terminal Zn-binding domains closely related to those of PolAlpha and PolEpsilonC. Collectively, the obtained results allowed us to propose a scenario for the evolution of eukaryotic PolBs.

Structural Analysis of Janus Kinases.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3863-3863
Author(s):  
Titus J. Boggon ◽  
Yiqun Li ◽  
Michael J. Eck
Keyword(s):  
Structural Information ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
Kinase Domain ◽  
Janus Kinase ◽  
Catalytically Active ◽  
Carboxy Terminal ◽  
Structural Insights ◽  
Kinase A

Abstract Janus Kinase 3 (Jak3) plays an essential role in hematopoietic signaling. Primarily expressed in B-, T- and Natural killer cells, it is activated through the γc chain of interleukin-2 like cytokine receptors (IL-2, -4, -7, -9, -15, -21). Deficiency of catalytically active Jak3 or disruption of the Jak3:IL-2γc interaction results in severe combined immunodeficiency (SCID). Jak3-deficient humans demonstrate defects restricted to the immune system, suggesting that selective inhibition of Jak3 catalytic activity or interruption of the Jak3:IL-2γc interaction are potentially exploitable strategies to achieve immunosuppression. Jak kinases contain four defined regions; a catalytically active carboxy-terminal kinase, a pseudo-kinase, a SH2-like region and a N-terminal Ferm domain. To date, no direct structural information has been reported for portions of any of the Jak family kinases. Structural studies are underway to define crystallographically the kinase domain of Jak3 and the Jak3-Ferm:IL-2γc interaction. Structural insights into the mechanism of Jak activation and routes to specific inhibition will be discussed.

Structural insights into the role of the Chl4–Iml3 complex in kinetochore assembly

2013 ◽  
Vol 69 (12) ◽  
pp. 2412-2419 ◽  
Author(s):  
Qiong Guo ◽  
Yuyong Tao ◽  
Hejun Liu ◽  
Maikun Teng ◽  
Xu Li
Keyword(s):  
De Novo ◽  
Molecular Level ◽  
Structural Information ◽  
Binding Activity ◽  
Intramolecular Interactions ◽  
Exact Role ◽  
Dna Binding Activity ◽  
Kinetochore Assembly ◽  
Structural Insights

Human CENP-N and CENP-L have been reported to selectively recognize the CENP-A nucleosome and to contribute to recruiting other constitutive centromere-associated network (CCAN) complexes involved in assembly of the inner kinetochore. As their homologues, Chl4 and Iml3 from budding yeast function in a similar way inde novoassembly of the kinetochore. A lack of biochemical and structural information precludes further understanding of their exact role at the molecular level. Here, the crystal structure of Iml3 is presented and the structure shows that Iml3 adopts an elongated conformation with a series of intramolecular interactions. Pull-down assays revealed that the C-terminal domain of Chl4, which forms a dimer in solution, is responsible for Iml3 binding. Acting as a heterodimer, the Chl4–Iml3 complex exhibits a low-affinity nonspecific DNA-binding activity which may play an important role in the kinetochore-assembly process.

scholarly journals The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

2007 ◽  
Vol 407 (3) ◽  
pp. 397-406 ◽  
Author(s):  
Victoria J. Just ◽  
Matthew R. Burrell ◽  
Laura Bowater ◽  
Iain McRobbie ◽  
Clare E. M. Stevenson ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Structural Information ◽  
Side Reaction ◽  
Oxalate Oxidase ◽  
Decarboxylase Activity ◽  
Oxalate Decarboxylase ◽  
Catalytically Active ◽  
The Stability ◽  
Kinetics Of

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural.

scholarly journals Mechanistic insights into a NOx storage-reduction (NSR) catalyst by spatiotemporal operando X-ray absorption spectroscopy

2019 ◽  
Vol 9 (5) ◽  
pp. 1103-1107
Author(s):  
Yasutaka Nagai ◽  
Akihiko Kato ◽  
Masaoki Iwasaki ◽  
Keisuke Kishita
Keyword(s):  
Absorption Spectroscopy ◽  
Mass Spectra ◽  
Active State ◽  
Nox Storage ◽  
Transient Phenomena ◽  
X Ray ◽  
Nox Storage Reduction ◽  
Catalytically Active ◽  
Fast Transient ◽  
X Ray Absorption

Monitoring the catalytically active state and online mass spectra clarified the fast transient phenomena occurring inside a NSR catalyst.

scholarly journals Exploring modular allostery via interchangeable regulatory domains

2018 ◽  
Vol 115 (12) ◽  
pp. 3006-3011 ◽  
Author(s):  
Yifei Fan ◽  
Penelope J. Cross ◽  
Geoffrey B. Jameson ◽  
Emily J. Parker
Keyword(s):  
Catalytic Domain ◽  
Chorismate Mutase ◽  
Ligand Specificity ◽  
Diverse Range ◽  
Regulatory Domains ◽  
Protein Functions ◽  
Catalytically Active ◽  
Allosteric Mechanisms ◽  
Common Architecture ◽  
New Protein

Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (β/α)8 catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These respective domains confer allosteric regulation by controlling DAH7PS function in response to ligand Tyr or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.

scholarly journals Simulating the function of sodium/proton antiporters

2015 ◽  
Vol 112 (40) ◽  
pp. 12378-12383 ◽  
Author(s):  
Raphael Alhadeff ◽  
Arieh Warshel
Keyword(s):  
Conformational Changes ◽  
Molecular Basis ◽  
Molecular Level ◽  
Structural Information ◽  
Coarse Grained ◽  
Charged State ◽  
Transport Models ◽  
Coarse Grained Model ◽  
Effective Transport ◽  
Molecular Origin

The molecular basis of the function of transporters is a problem of significant importance, and the emerging structural information has not yet been converted to a full understanding of the corresponding function. This work explores the molecular origin of the function of the bacterial Na+/H+ antiporter NhaA by evaluating the energetics of the Na+ and H+ movement and then using the resulting landscape in Monte Carlo simulations that examine two transport models and explore which model can reproduce the relevant experimental results. The simulations reproduce the observed transport features by a relatively simple model that relates the protein structure to its transporting function. Focusing on the two key aspartic acid residues of NhaA, D163 and D164, shows that the fully charged state acts as an Na+ trap and that the fully protonated one poses an energetic barrier that blocks the transport of Na+. By alternating between the former and latter states, mediated by the partially protonated protein, protons, and Na+ can be exchanged across the membrane at 2:1 stoichiometry. Our study provides a numerical validation of the need of large conformational changes for effective transport. Furthermore, we also yield a reasonable explanation for the observation that some mammalian transporters have 1:1 stoichiometry. The present coarse-grained model can provide a general way for exploring the function of transporters on a molecular level.

Unified structural motifs of the catalytically active state of Co(oxyhydr)oxides during the electrochemical oxygen evolution reaction

2018 ◽  
Vol 1 (9) ◽  
pp. 711-719 ◽  
Author(s):  
Arno Bergmann ◽  
Travis E. Jones ◽  
Elias Martinez Moreno ◽  
Detre Teschner ◽  
Petko Chernev ◽  
...  
Keyword(s):  
Oxygen Evolution ◽  
Oxygen Evolution Reaction ◽  
Active State ◽  
Structural Motifs ◽  
Catalytically Active ◽  
Electrochemical Oxygen

scholarly journals Crystal Structures of the Receiver Domain of the Response Regulator PhoP from Escherichia coli in the Absence and Presence of the Phosphoryl Analog Beryllofluoride

2007 ◽  
Vol 189 (16) ◽  
pp. 5987-5995 ◽  
Author(s):  
Priti Bachhawat ◽  
Ann M. Stock
Keyword(s):  
Escherichia Coli ◽  
Crystal Structures ◽  
Response Regulator ◽  
Active State ◽  
Two Component System ◽  
Component System ◽  
Regulatory Domains ◽  
Receiver Domain ◽  
Two Component ◽  
High Concentrations

ABSTRACT The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg2+. Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation.

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