Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβ in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ2) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ2, and Pαβγ2–cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and β-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.

Phosphodiesterase 6 (PDE6) is central for visual transduction in rod and cone cells. PDE6 is a heterodimeric enzyme consisting of α and β catalytic subunits (Pαβ) to which two identical inhibitory subunits (Pγ) are bound (Pαβγγ). Light activation of PDE6 results from transducin binding to Pγ and relieving inhibition of catalysis. Previous studies have shown two different classes of Pγ binding sites on Pαβ, but the structural basis for the differences in binding affinity of Pγ is unknown. We hypothesized that Pγ preferentially binds to one catalytic subunit due to differences in the amino acid sequence of the two subunits. To test this, we used chemical cross‐linking of Pγ to Pαβ followed by mass spectrometric identification of which catalytic subunit was cross‐linked over a range of Pγ concentrations. Even at sub‐stoichiometric levels of Pγ relative to Pαβ, we failed to observe a statistically significant difference in Pγ binding. This lack of preferential interaction of Pγ with the catalytic subunits at sub‐stoichiometric levels suggests that binding of the first Pγ subunit allosterically reduces the affinity with which the second Pγ subunit binds to the Pαβ catalytic dimer. This may also account for biochemical evidence that transducin preferentially interacts with one of the catalytic subunits during light activation. Supported by NIH grant EY‐05798 (RHC) and NH Agricultural Experiment Station grant NH00567 (FC).

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme of visual signaling. Its activity is regulated by the inhibitory subunit (Pγ). Upon light stimulation, activated transducin (Tα*) relieves Pγ inhibition of the PDE6 catalytic dimer (Pαβ), activates PDE6, and causes a drop in cellular cGMP levels. However, the mechanism by which Tα* activates PDE6 is unclear. Although structures for some PDEs have been solved, no high‐resolution structures of PDE6 are available. Using homology modeling, we generated models of the GAF and catalytic domains of the α and β catalytic subunits. Using PDE2 (PDB:3IBJ) as a template, we built a topology model for the full‐length Pαβ catalytic dimer. Chemical cross‐linking of Pαβ identified 18 cross‐links that validated our homology model. We also identified multiple cross‐links between Pγ and Pαβ that support an extended structure for Pγ bound to Pαβ, as well as sites within the N‐terminal region of Pγ which may allosterically regulate cGMP binding to PDE6 GAF domains. Preliminary cross‐linking results of Tα*‐activated PDE6 reveal multiple sites of interaction of Tα* with Pα, Pβ, and Pγ. We conclude that cross‐linking and molecular modeling provide unique insights into PDE6 regulation not available by other approaches. Supported by NIH grant EY‐05798 (RHC), NH Agricultural Experiment Station grant NH00567 (FC), and the Karabelas Faculty Development Fund (FC).

The cGMP phosphodiesterase (PDE6) involved in visual transduction in photoreceptor cells contains two inhibitory gamma-subunits (P gamma) which bind to the catalytic core (P alpha beta) to inhibit catalysis and stimulate cGMP binding to the GAF domains of P alpha beta. During visual excitation, interaction of activated transducin with P gamma relieves inhibition. P gamma also participates in a complex with RGS9-1 and other proteins to accelerate the GTPase activity of activated transducin. We studied the structural determinants for these important functions of P gamma. First, we identified two important sites in the middle region of P gamma (amino acids 27-38 and 52-54) that significantly stabilize the overall binding affinity of P gamma with P alpha beta. The ability of P gamma to stimulate noncatalytic cGMP binding to the GAF domains of PDE6 has been localized to amino acids 27-30 of P gamma. Transducin activation of PDE6 catalysis critically depends on the presence of Ile54 in the glycine-rich region of P gamma in order to relieve inhibition of catalysis. The central glycine-rich region of P gamma is also required for transducin to increase cGMP exchange at the GAF domains. Finally, Thr-65 and/or Val-66 of P gamma are critical residues for P gamma to stimulate GTPase activity of transducin in a complex with RGS9-1. We propose that the glycine-rich region of P gamma is a primary docking site for PDE6-interacting proteins involved in the activation/inactivation pathways of visual transduction. This functional mapping of P gamma with its binding partners demonstrates the remarkable versatility of this multifunctional protein and its central role in regulating the activation and lifetime of visual transduction.

The SMU.2055 gene from the major caries pathogen Streptococcus mutans is annotated as a putative acetyltransferase with 163 amino-acid residues. In order to identify its function via structural studies, the SMU.2055 gene was cloned into the expression vector pET28a. Native and SeMet-labelled SMU.2055 proteins with a His(6) tag at the N-terminus were expressed at a high level in Escherichia coli strain BL21 (DE3) and purified to homogeneity by Ni(2+)-chelating affinity chromatography. Diffraction-quality crystals of SeMet-labelled SMU.2055 were obtained using the sitting-drop vapour-diffusion method and diffracted to a resolution of 2.5 A on beamline BL17A at the Photon Factory, Tsukuba, Japan. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 92.0, b = 95.0, c = 192.2 A. The asymmetric unit contained four molecules, with a solvent content of 57.1%.

Expression,purification and biochemical characterization of N-acetylglutamate kinase gene(argB) from Streptococcus mutans,the leading pathogen of human dental caries,were studied.The argB gene was amplified by polymerase chain reaction(PCR) with S.mutans genomic DNA as template.The PCR fragments were digested and ligated into the cloning/expression vector pET28a to construct the recombinant pET28a-argB.After verified by DNA sequencing,it was transformed into expressing cells E.coli BL21(DE3).The SDS-PAGE assay on the expressing cells E.coli BL21(DE3)/pET28a-argB displayed that the argB gene could be expressed in soluable form after induction with IPTG.The target NAGK was obtained by Ni chelating chromatography and gel size-exclusion chromatography.Enzyme activity assays conformed that the key enzyme S.mutans NAGK isn't inhibited by arginine,the final product in the acetyl cyclic route of arginine biosynthesis;thus implied that there may be other regulation mechanism,so it was necessary to make further investigation into arginine biosynthesis in S.mutans.The observation of analytical gel chromatography demonstrated that the enzymatic S.mutans NAGK is monomeric,apparently which was inconsistent with the reports about that in the amino acid kinase family.

The N-acetylglutamate kinase from Streptococcus mutans was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.06 A. The crystal belonged to space group P2(1)2(1)2, with unit cell parameters a = 57.19 A, b =94.76 A, c =47.58 A. The gel filtration and initial phasing results showed that the enzyme exists as a monomer, which is different from previously reported N-acetylglutamate kinases.

The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A, beta = 98.82 degrees.

The smu .961 gene encodes a putative protein of 183 residues in Streptococcus mutans , a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A˚ resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C 2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A˚, (cid:1) = 98.82 (cid:1) .