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Biochemical and detailed structural information of human ether-ago- go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies. [ABSTRACT FROM AUTHOR]

2,3′-Cyclic nucleotide-3′-phosphodiesterase (CNP) is a myelin-associated protein, an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2′,3′-cyclic nucleotides to produce 2′-nucleotides, but the physiologically relevant in vivo substrate is still unknown. Recently, it was found that CNP is a possible linker protein between microtubules and the plasma membranes. Since CNP is modified post-translationally by an isoprenylation process at its C terminus, the prenylation is hypothesized to be a requisite process, which permanently anchors CNP to the plasma membrane. This study investigates the molecular mechanism of the interaction between CNP and the plasma membrane, proposing a general model to interpret the structural bases of prenylated proteins binding to the membrane. A 13 residue, C-terminal CNP fragment, C13, was demonstrated to be directly responsible for CNP membrane anchoring. C13 and its lipidated derivative (LIPO-C13) were subjected to conformational analysis in membrane mimetic environments, by means of CD and NMR spectroscopies. The orientation of C13 in relation to the membrane was investigated by NMR and EPR spin labeling studies. Our structural investigation shows that the presence of the lipidic tail is essential for the peptide to be folded and correctly positioned on the membrane surface. A general model is proposed in which the post-translational lipidation is an important biomolecular trick to enlarge the hydrophobic surface and to enable the contact of the protein with membrane. [ABSTRACT FROM AUTHOR]

The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient. [ABSTRACT FROM AUTHOR]

Photoreceptor rod and cone phosphodiesterases comprise the sixth family of cyclic nucleotide phosphodiesterases (PDE6). PDE6s have uniquely evolved as effector enzymes in the vertebrate phototransduction cascade. To understand the evolution of the PDE6 family, we have examined PDE6 in lamprey, an ancient vertebrate group. A single PDE6 catalytic subunit transcript was found in the sea lamprey Petromyzon marinus cDNA library. The lamprey PDE6 sequence showed a high degree of homology with mammalian PDE6 and equally distant relationships with the rod and cone enzymes. In contrast, two different PDE6 inhibitory Pγ subunits, a cone-type Pγ 1 and a mixed cone/rod-type Pγ2, have been identified in the lamprey retina. Immunofluorescence analysis demonstrated that Pγ1 and Pγ2 are expressed in the long and short photoreceptors of sea lamprey, respectively. The catalytic PDE6 subunit was present in the photoreceptors of both types and colocalized with the Pγ subunits. Recombinant Pγ1 and Pγ2 potently inhibited trypsin-activated lamprey and bovine PDE6 enzymes. Our results point to a high degree of conservation of PDE6 genes during the vertebrate evolution. The apparent duplication of the Pγ gene in the stem of vertebrate lineage may have been an essential component of the evolution of scotopic vision in early vertebrates. [ABSTRACT FROM AUTHOR]

Phosphodiesterases (PDEs) are enzymes that modulate cyclic nucleotide signaling and as such are clinical targets for a range of disorders including congestive heart failure, erectile dysfunction, and inflammation. The PDE3 family comprises two highly homologous subtypes expressed in different tissues, and inhibitors of this family have been shown to increase lipolysis in adipocytes. A specific PDE3B (the lipocyte-localized subtype) inhibitor would be a very useful tool to evaluate the effects of PDE3 inhibition on lipolysis and metabolic rate and might become a novel tool for treatment of obesity. We report here the three-dimensional structures of the catalytic domain of human PDE3B in complex with a generic PDE inhibitor and a novel PDE3 selective inhibitor. These structures explain the dual cAMP/cGMP binding capabilities of PDE3, provide the molecular basis for inhibitor specificity, and can supply a valid platform for the design of improved compounds. [ABSTRACT FROM AUTHOR]

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations of cyclic 3',5'-adenosine and guanosine monophosphates (cAMP and cGMP, respectively) by hydrolyzing them to AMP and GMP, respectively. Family-selective inhibitors of PDEs have been studied for treatment of various human diseases. However, the catalytic mechanism of cyclic nucleotide hydrolysis by PDEs has remained unclear. We determined the crystal structure of the human PDE4D2 catalytic domain in complex with AMP at 2.4 Å resolution. In this structure, two divalent metal ions simultaneously interact with the phosphate group of AMP, implying a binuclear catalysis. In addition, the structure suggested that a hydroxide ion or a water bridging two metal ions may serve as the nucleophile for the hydrolysis of the cAMP phosphodiester bond. [ABSTRACT FROM AUTHOR]

Shows that mutation of either His-607 or His-643 to alanine profoundly diminishes support of cyclic nucleotide phosphodiesterase (PDE) catalysis by Mn2+ or Mg2+. Residues on cGMP-binding cGMP-specific PDE that contribute to interactions with catalytically relevant metals; Use of a distinct complement of residues for interacting with metals.