Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the gamma-subunit.

ABSTRACT: The inhibitory interaction of phosphodiesterase-6 (PDE6) with its gamma-subunit (Pgamma) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pgamma-inhibitory peptide Pgamma(70-87) have been determined at 2.9 and 3.0 A, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pgamma and suggest the conformational change of Pgamma on transducin activation. Two variable H- and M-loops of PDE5/6cd form a distinct interface that contributes to the Pgamma-binding site. This allows the Pgamma C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pgamma-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pgamma(70-87)-binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

SUBMITTER: Barren B

PROVIDER: S-EPMC2782096 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Determinants for phosphodiesterase 6 inhibition by its gamma-subunit.

Project description:The interaction of phosphodiesterase 6 (PDE6) with its inhibitory Pgamma-subunits (Pgamma) is unparalleled among PDE families and is central to vertebrate phototransduction. The C-terminus of Pgamma occludes the active site of PDE6, thereby preventing hydrolysis of cGMP. In this study, we examine the determinants of this critical interaction using structure-based loss-of-function mutagenesis of a chimeric PDE5/PDE6 catalytic domain and gain-of-function mutagenesis of the PDE5 catalytic domain. This analysis revealed the key role of PDE6-specific residues within the catalytic domain M-loop-alpha-helix 15 region and suggested an important contribution of the H-loop-M-loop interface to the PDE6 inhibition by the Pgamma C-terminus. Identification of the determinants for the PDE6-Pgamma interaction offers insights into the evolution of the visual effector enzyme.

| S-EPMC2864356 | biostudies-literature

Structural requirements of the photoreceptor phosphodiesterase gamma-subunit for inhibition of rod PDE6 holoenzyme and for its activation by transducin.

Project description:The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its gamma-subunit (Pgamma), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pgamma are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pgamma78-87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pgamma C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pgamma inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact alpha2 helix of Pgamma stabilize binding of C-terminal Pgamma peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pgamma molecule and tested for activation by transducin, we found that the C-terminal region (Pgamma63-87) by itself could not be displaced but that transducin could relieve inhibition of certain Pgamma truncation mutants. Our results are consistent with two distinct mechanisms of Pgamma inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pgamma to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pgamma to effectively relieve its inhibitory constraint.

| S-EPMC2836051 | biostudies-literature

N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex.

Project description:In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.

| S-EPMC3083170 | biostudies-literature

Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit.

Project description:Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.

| S-EPMC5571044 | biostudies-literature

Structural basis of ribosomal 30S subunit degradation by RNase R

Project description:Protein synthesis is a major energy-consuming process of the cell, which requires controlled production and turnover of ribosomes. While the last years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3’ to 5’ exonuclease, RNase R. The structures reveal that RNase R binds initially to the 30S platform to facilitate degradation of the functionally important anti-Shine-Dalgarno sequence and decoding site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S, which is overcome by a major structural rearrangement of the 30S head, involving loss of ribosomal proteins. RNase R parallels this movement, relocating to the decoding site, by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also that the enzyme alone is sufficient for complete 30S degradation. Collectively, our results provide a mechanistic basis for RNase R-mediated 30S degradation and reveal that RNase R targets orphaned 30S subunits using a dynamic anchored binding site switching mechanism.

2023-12-25 | GSE251701 | GEO

Transducin activates cGMP phosphodiesterase by trapping inhibitory γ subunit freed reversibly from the catalytic subunit in solution.

Project description:Activation of cGMP phosphodiesterase (PDE) by activated transducin α subunit (Tα*) is a necessary step to generate a light response in vertebrate photoreceptors. PDE in rods is a heterotetramer composed of two catalytic subunits, PDEα and PDEβ, and two inhibitory PDEγ subunits, each binding to PDEα or PDEβ. Activation of PDE is achieved by relief of the inhibitory constraint of PDEγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of Tα* to PDEγ still bound to PDEα or PDEβ seems to be difficult because the binding site of PDEγ to PDEα or PDEβ overlaps with the binding site to Tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates PDE by trapping PDEγ released reversibly and spontaneously from the catalytic subunit. This mechanism well explains PDE activation by Tα* in solution. Our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment.

| S-EPMC6510727 | biostudies-literature

Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin alpha-subunit.

Project description:In the present report, the region of interaction between the GDP-bound alpha-subunit of transducin (alphat.GTP) and the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) has been studied. It is widely accepted that the alphat.GTP is the active form of transducin and that the GDP-bound transducin alpha-subunit (alphat. GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pgamma on alphat.GTP is in a region between the exposed face of the alpha3 and alpha4 helices of alphat.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900-26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP)-derivatized Pgamma (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pgamma to alphat.GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound transducin alpha-subunit (alphat-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68-->Ala Pgamma mutant, and by pretreatment of the alphat.GDP with the betagamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pgamma specifically transferred the [125I]APC group to Cys-250 and Cys-210 of alphat.GDP. These data indicate that the C-terminal region (especially Cys-68-Trp-70) of Pgamma interacts with alphat. GDP on the exposed interface between alpha2/beta4 and alpha3/beta5 of the alpha-subunit of transducin. Disulphide exchange was also observed with the alpha-subunit of holotransducin but this was only approx. 60% of that of pure alphat.GDP. The variation in the binding pattern between alphat.GDP and alphat.GTP with the C-terminal region of Pgamma may contribute to the functional difference between the GDP- and GTP-bound states.

| S-EPMC1219963 | biostudies-other

Structural Basis of the Inhibition of L-Methionine γ-Lyase from <i>Fusobacterium nucleatum</i>.

Project description:Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5'-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

| S-EPMC9865163 | biostudies-literature

Structural basis for the design of selective phosphodiesterase 4B inhibitors.

Project description:Phosphodiesterase-4B (PDE4B) regulates the pro-inflammatory Toll Receptor -Tumor Necrosis Factor ? (TNF?) pathway in monocytes, macrophages and microglial cells. As such, it is an important, although under-exploited molecular target for anti-inflammatory drugs. This is due in part to the difficulty of developing selective PDE4B inhibitors as the amino acid sequence of the PDE4 active site is identical in all PDE4 subtypes (PDE4A-D). We show that highly selective PDE4B inhibitors can be designed by exploiting sequence differences outside the active site. Specifically, PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix, now termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP. PDE4B selectivity is driven by a single amino acid polymorphism in CR3 (Leu674 in PDE4B1 versus Gln594 in PDE4D). The reciprocal mutations in PDE4B and PDE4D cause a 70-80 fold shift in selectivity. Our structural studies show that CR3 is flexible and can adopt multiple orientations and multiple registries in the closed conformation. The new co-crystal structure with bound ligand provides a guide map for the design of PDE4B selective anti-inflammatory drugs.

| S-EPMC4057648 | biostudies-literature

Structural basis for the catalytic mechanism of human phosphodiesterase 9.

Project description:The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.

| S-EPMC2533186 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data