scholarly journals Determinants of ligand binding and catalytic activity in the myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Arne Raasakka ◽  
Matti Myllykoski ◽  
Saara Laulumaa ◽  
Mari Lehtimäki ◽  
Michael Härtlein ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Ligand Binding ◽  
Cyclic Nucleotide

Binding of cyclic nucleotides to phosphodiesterase 10A and 11A GAF domains does not stimulate catalytic activity

2009 ◽  
Vol 423 (3) ◽  
pp. 401-409 ◽  
Author(s):  
Karina Matthiesen ◽  
Jacob Nielsen
Keyword(s):  
Catalytic Activity ◽  
Ligand Binding ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Terminal Part ◽  
Indirect Assessment ◽  
High Affinity ◽  
Catalytic Sites ◽  
Phosphodiesterase 10A ◽  
Camp And Cgmp

To date eleven human PDE (3′,5′-cyclic nucleotide phosphodiesterase) families have been identified. Of these, five families contain non-catalytic tandem GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaenaadenylate cyclases and Escherichia coliFhlA) domains, GAFa and GAFb, in the N-terminal part of the enzyme. For PDE2A, PDE5A and PDE6 the GAF domains have been shown to bind cGMP with high affinity. For PDE2A and PDE5A this ligand binding has been shown to stimulate the catalytic activity of the enzyme. PDE10A and PDE11A are the two most recently described PDEs and it has been suggested that their GAF domains bind to cAMP and cGMP respectively. We have developed a scintillation proximity-based assay to directly measure cyclic nucleotide binding to the PDE2A, PDE10A and PDE11A GAF domains, and in the present study we demonstrate binding of cyclic nucleotides to the PDE10A and PDE11A GAF domains. We show that these non-catalytic sites bind cAMP and cGMP respectively with much higher affinity than has previously been suggested using indirect assessment of the interaction. The GAFb domain of PDE10A binds cAMP with a Kd of 48 nM and the GAFa domain of PDE11A binds cGMP with a Kd of 110 nM. The effect of cyclic nucleotides binding to the GAF domains on the enzyme activity was investigated through the use of modified cyclic nucleotides. In contrast with other GAF domain-containing PDEs, and with what has previously been predicted, ligand binding to the GAF domains of PDE10A and PDE11A does not stimulate catalytic activity.

scholarly journals Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e32336 ◽  
Author(s):  
Matti Myllykoski ◽  
Arne Raasakka ◽  
Huijong Han ◽  
Petri Kursula
Keyword(s):  
Ligand Binding ◽  
Active Site ◽  
Cyclic Nucleotide ◽  
Molecular Conformation

scholarly journals Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

2012 ◽  
Vol 140 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Shengjun Wu ◽  
Weihua Gao ◽  
Changan Xie ◽  
Xinping Xu ◽  
Christina Vorvis ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Domain ◽  
Hcn Channels ◽  
Channel Blockers ◽  
Structural Elements ◽  
Hcn Channel ◽  
Channel Interaction ◽  
State Dependent ◽  
Activation Gate

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide–gated (CNG) and hyperpolarization-activated, cyclic nucleotide–regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand–channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand–whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs+ and Mg2+, effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

scholarly journals Graph Theoretical Methods and Workflows for Searching and Annotation of RNA Tertiary Base Motifs and Substructures

2021 ◽  
Vol 22 (16) ◽  
pp. 8553
Author(s):  
Reeki Emrizal ◽  
Hazrina Yusof Hamdani ◽  
Mohd Firdaus-Raih
Keyword(s):  
Functional Analysis ◽  
Hydrogen Bond ◽  
Catalytic Activity ◽  
Ligand Binding ◽  
Protein Data Bank ◽  
Structural Stability ◽  
Data Bank ◽  
Computational Approaches ◽  
Theoretical Methods ◽  
Structure Annotation

The increasing number and complexity of structures containing RNA chains in the Protein Data Bank (PDB) have led to the need for automated structure annotation methods to replace or complement expert visual curation. This is especially true when searching for tertiary base motifs and substructures. Such base arrangements and motifs have diverse roles that range from contributions to structural stability to more direct involvement in the molecule’s functions, such as the sites for ligand binding and catalytic activity. We review the utility of computational approaches in annotating RNA tertiary base motifs in a dataset of PDB structures, particularly the use of graph theoretical algorithms that can search for such base motifs and annotate them or find and annotate clusters of hydrogen-bond-connected bases. We also demonstrate how such graph theoretical algorithms can be integrated into a workflow that allows for functional analysis and comparisons of base arrangements and sub-structures, such as those involved in ligand binding. The capacity to carry out such automatic curations has led to the discovery of novel motifs and can give new context to known motifs as well as enable the rapid compilation of RNA 3D motifs into a database.

scholarly journals Quantifying the cooperative subunit action in a multimeric membrane receptor

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nisa Wongsamitkul ◽  
Vasilica Nache ◽  
Thomas Eick ◽  
Sabine Hummert ◽  
Eckhard Schulz ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Sites ◽  
Membrane Receptor ◽  
Membrane Receptors ◽  
Cooperative Action ◽  
The Individual ◽  
Insight Into

Abstract In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.

scholarly journals Constraining Ligand-Binding Site Stoichiometry Suggests that a Cyclic Nucleotide–Gated Channel Is Composed of Two Functional Dimers

Neuron ◽  
1998 ◽  
Vol 21 (1) ◽  
pp. 235-248 ◽  
Author(s):  
David T. Liu ◽  
Gareth R. Tibbs ◽  
Pierre Paoletti ◽  
Steven A. Siegelbaum
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Cyclic Nucleotide ◽  
Ligand Binding Site ◽  
Gated Channel

scholarly journals Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase in Human Cerebral Cortex and Glioblastoma Multiforme

1996 ◽  
Vol 23 (4) ◽  
pp. 245-250 ◽  
Author(s):  
Sumeer Lal ◽  
Rajala V.S. Raju ◽  
Robert J.B. Macaulay ◽  
Rajendra K. Sharma
Keyword(s):  
Cerebral Cortex ◽  
Catalytic Activity ◽  
Glioblastoma Multiforme ◽  
Brain Tumors ◽  
Polyclonal Antibody ◽  
Spectrophotometric Method ◽  
Cyclic Nucleotide ◽  
Stereotactic Biopsy ◽  
Bovine Brain ◽  
Cerebral Tissue

AbstractBackground: Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) has been extensively studied and characterized in normal mammalian tissues; however very little is known about this enzyme in human brain tumors. It has been established that high levels of this enzyme exist in non-central nervous system tumors, PDE inhibitors or cAMP analogues have been used to treat them. This study has examined the levels of CaMPDE in glioblastoma multiforme from six patients and has compared these to the levels of CaMPDE in four patients with normal cerebral tissue. In addition, an enzyme immune assay method (EIA) was developed in this study for the detection of CaMPDE in human cerebral tissue. This method is proposed to be used as an adjunct to the spectrophotometric method presently utilized. This would be beneficial in cases where small tissue samples, for example in stereotactic biopsy, are available. Methods: The CaMPDE activity and corresponding levels of expression in cerebral tissue from temporal lobectomies and both surgical extraction or stereotactic biopsy in patients with primary tumors were determined by spectophotometric and EIA, respectively. The EIA was developed from the production of a polyclonal antibody against bovine brain 60 kDa CaMPDE isozyme. Cross reactivity of the antibody with human was confirmed using transblot and immunohistochemistry. Results: Utilising the EIA, there was found to be significant reduction in both catalytic activity (p < 0.001) and in quantitative protein expression (p < 0.001) in glioblastoma multiforme from patients when compared to normal cerebral cortex. Immunoblotting experiments and immunohistochemistry demonstrated that CaMPDE in glioblastoma multiforme failed to react with a polyclonal antibody raised against bovine brain 60 kDa CaMPDE isozyme, whereas the enzyme from normal tissue reacted with antibody. Conclusions: Contrary to other studies on non-CNS tumors, the catalytic activity and the protein expression of CaMPDE is reduced in glioblastoma multiforme. The EIA method is a more sensitive in detecting CaMPDE than in the spectrophotometric method, especially when a small amount of tissue is available. Immunohistochemistry and the EIA may be useful in the future to use as markers for other types of brain tumors and not for glioblastoma multiforme as demonstrated.

scholarly journals Activity and Affinity of Pin1 Variants

2019 ◽  
Author(s):  
Alexandra Born ◽  
Morkos A. Henen ◽  
Beat Vögeli
Keyword(s):  
Catalytic Activity ◽  
Ligand Binding ◽  
Binding Site ◽  
Structure And Function ◽  
Ligand Binding Site ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity ◽  
Ppiase Domain ◽  
And Function

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1&rsquo;s structure and function.

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