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Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase.

ABSTRACT: The 2H phosphoesterase family contains enzymes with two His-X-Ser/Thr motifs in the active site. 2H enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. For many 2H enzymes, the physiological function is unknown. Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2'-AMP, 3'-AMP, phosphate, and NADP+. Comparisons to the well-characterized vertebrate myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) highlight specific features of the catalytic cycle and substrate recognition in both enzymes. The role played by the helix ?7, unique to CNPases within the 2H family, is apparently taken over by Arg130 in the bacterial enzyme. Other residues and loops lining the active site groove are likely to be important for RNA substrate binding. We visualized conformational changes related to ligand binding, as well as the position of the nucleophilic water molecule. We also present a low-resolution model of E. coli LigT bound to tRNA in solution, and provide a model for RNA binding by LigT, involving flexible loops lining the active site cavity. Taken together, our results both aid in understanding the common features of 2H family enzymes and help highlight the distinct features in the 2H family members, which must result in different reaction mechanisms. Unique aspects in different 2H family members can be observed in ligand recognition and binding, and in the coordination of the nucleophilic water molecule and the reactive phosphate moiety.

SUBMITTER: Myllykoski M

PROVIDER: S-EPMC5283653 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase.

Myllykoski Matti M Kursula Petri P

PloS one 20170131 1

The 2H phosphoesterase family contains enzymes with two His-X-Ser/Thr motifs in the active site. 2H enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. For many 2H enzymes, the physiological function is unknown. Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2'-AMP, 3'-AMP, phosphate, and NADP+. Compar ...[more]

PMID: 28141848

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Project description:Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

Project description:2',3' Cyclic nucleotide phosphodiesterases are enzymes that catalyze at least two distinct steps in the splicing of tRNA introns in eukaryotes. Recently, the biochemistry and structure of these enzymes, from yeast and the plant Arabidopsis thaliana, have been extensively studied. They were found to share a common active site, characterized by two conserved histidines, with the bacterial tRNA-ligating enzyme LigT and the vertebrate myelin-associated 2',3' phosphodiesterases. Using sensitive sequence profile analysis methods, we show that these enzymes define a large superfamily of predicted phosphoesterases with two conserved histidines (hence 2H phosphoesterase superfamily). We identify several new families of 2H phosphoesterases and present a complete evolutionary classification of this superfamily. We also carry out a structure- function analysis of these proteins and present evidence for diverse interactions for different families, within this superfamily, with RNA substrates and protein partners. In particular, we show that eukaryotes contain two ancient families of these proteins that might be involved in RNA processing, transcriptional co-activation and post-transcriptional gene silencing. Another eukaryotic family restricted to vertebrates and insects is combined with UBA and SH3 domains suggesting a role in signal transduction. We detect these phosphoesterase modules in polyproteins of certain retroviruses, rotaviruses and coronaviruses, where they could function in capping and processing of viral RNAs. Furthermore, we present evidence for multiple families of 2H phosphoesterases in bacteria, which might be involved in the processing of small molecules with the 2',3' cyclic phosphoester linkages. The evolutionary analysis suggests that the 2H domain emerged through a duplication of a simple structural unit containing a single catalytic histidine prior to the last common ancestor of all life forms. Initially, this domain appears to have been involved in RNA processing and it appears to have been recruited to perform various other functions in later stages of evolution.

Dataset Information

Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase.

Publications

Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase.

Similar Datasets

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