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. 2014 Nov 11;426(22):3713-3728.
doi: 10.1016/j.jmb.2014.07.033. Epub 2014 Aug 19.

Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling

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Free PMC article

Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling

Xiaohui Zeng-Elmore et al. J Mol Biol. .
Free PMC article

Abstract

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.

Keywords: chemical cross-linking; integrative modeling; mass spectrometry; phosphodiesterase 6 (PDE6); visual transduction.

Figures

Figure 1
Figure 1
Chemical cross-linking of the PDE6 holoenzyme. A) Purified PDE6 holoenzyme or the Pαβ catalytic dimer reconstituted with either wild-type (Pγ) or a quadruple lysine site-directed mutant of Pγ (Pγ4K) were cross-linked with BS3 or Sulfo-MBS. Control samples (Ctrl) were treated identically except that cross-linkers were omitted. Gel bands of uncross-linked subunits (γ and α/β) and cross-linked subunits (αγ or βγ at ~110 kD; αβ or αβγ or αβγγ at ~220 kD) are indicated. B) Tandem MS spectrum of a cross-linked peptide (at m/z 1004.213+) from the 220 kD band, with sequence fragments from both α-(K447-K455) and β-subunit (S612-K627). Sequence ion series from both peptide moieties identify the cross-link between Pα K447 and Pβ K618. Fragments with upper case labels contain the cross-linked peptide moiety. C) Differential LC-MS analysis to determine the cross-linked peptide shown in Fig. 1B (at m/z 1004.213+) as an intersubunit cross-link. D) Superimposition of a comparative model for Pβ catalytic domain(blue) with the structure of chimeric PDE5/PDE6 catalytic domain (gray; PDB: 3JWR). H-loops are colored in green; M-loops and the α-helix 15 regions are colored in cyan. The C-terminal segment of Pγ in the PDE5/PDE6 chimera structure is colored in orange. Cross-linked residues between Pγ and Pβ that can be mapped in the structure are indicated as magenta spheres.
Figure 1
Figure 1
Chemical cross-linking of the PDE6 holoenzyme. A) Purified PDE6 holoenzyme or the Pαβ catalytic dimer reconstituted with either wild-type (Pγ) or a quadruple lysine site-directed mutant of Pγ (Pγ4K) were cross-linked with BS3 or Sulfo-MBS. Control samples (Ctrl) were treated identically except that cross-linkers were omitted. Gel bands of uncross-linked subunits (γ and α/β) and cross-linked subunits (αγ or βγ at ~110 kD; αβ or αβγ or αβγγ at ~220 kD) are indicated. B) Tandem MS spectrum of a cross-linked peptide (at m/z 1004.213+) from the 220 kD band, with sequence fragments from both α-(K447-K455) and β-subunit (S612-K627). Sequence ion series from both peptide moieties identify the cross-link between Pα K447 and Pβ K618. Fragments with upper case labels contain the cross-linked peptide moiety. C) Differential LC-MS analysis to determine the cross-linked peptide shown in Fig. 1B (at m/z 1004.213+) as an intersubunit cross-link. D) Superimposition of a comparative model for Pβ catalytic domain(blue) with the structure of chimeric PDE5/PDE6 catalytic domain (gray; PDB: 3JWR). H-loops are colored in green; M-loops and the α-helix 15 regions are colored in cyan. The C-terminal segment of Pγ in the PDE5/PDE6 chimera structure is colored in orange. Cross-linked residues between Pγ and Pβ that can be mapped in the structure are indicated as magenta spheres.
Figure 2
Figure 2
Modeling of the PDE6 catalytic dimer. A) Homology model of the PDE6 catalytic dimer based on multiple template structures. B) A representative of the most populated cluster of Pαβ heterodimer models produced by the integrative modeling approach and structure refinement. The fit of PDE6 model in its EM molecular density map (12) is shown. Bottom images are of the catalytic domain viewed along the long axis of the catalytic dimer. H-loops are colored in green; cross-links are represented by red solid lines. The Pα and Pβ chains are represented by blue and light brown ribbons, respectively.
Figure 3
Figure 3
Cross-linking preference of Pγ K25 for the α-subunit. A) Tandem MS spectrum of a Pγ peptide containing K25 cross-linked with Sulfo-MBS to a Pα peptide containing C163. B) Structural model of the GAFa domains of Pα (blue) and Pβ (light brown). C163 on the α–subunit is in close proximity to the ‘lid’ region of the noncatalytic cGMP (ball and stick atoms) binding site. Below is the sequence alignment of the bovine Pα and Pβ. C) MS analysis detected the unmodified tryptic peptides from both Pα (containing C163) and Pβ (containing C157). D) The reduction of Pβ in the Sulfo-MBS cross-linked 110 kD band, and the increase of Pβ in the EDC cross-linked 110 kD band. The number of unique peptide spectra (sequence discriminates Pα from Pβ; gray) as well as the number of total peptide spectra (black) for Pα and Pβ were determined to calculate the Pβ/Pα ratio. Statistical significance was evaluated by ANOVA analysis using Tukey’s test (for 110 kD samples annotated with asterisks).
Figure 3
Figure 3
Cross-linking preference of Pγ K25 for the α-subunit. A) Tandem MS spectrum of a Pγ peptide containing K25 cross-linked with Sulfo-MBS to a Pα peptide containing C163. B) Structural model of the GAFa domains of Pα (blue) and Pβ (light brown). C163 on the α–subunit is in close proximity to the ‘lid’ region of the noncatalytic cGMP (ball and stick atoms) binding site. Below is the sequence alignment of the bovine Pα and Pβ. C) MS analysis detected the unmodified tryptic peptides from both Pα (containing C163) and Pβ (containing C157). D) The reduction of Pβ in the Sulfo-MBS cross-linked 110 kD band, and the increase of Pβ in the EDC cross-linked 110 kD band. The number of unique peptide spectra (sequence discriminates Pα from Pβ; gray) as well as the number of total peptide spectra (black) for Pα and Pβ were determined to calculate the Pβ/Pα ratio. Statistical significance was evaluated by ANOVA analysis using Tukey’s test (for 110 kD samples annotated with asterisks).
Figure 4
Figure 4
Photoactivatable probes on Pγ reveal preferential interactions of Pγ with the α- or β-subunit of PDE6. A) Tandem MS spectrum reveals the cross-link between Pγ23Bpa and Pα Phe165. B) Pβ/Pα ratio for the number of peptide spectra identified in the 110 kD gel band with Pγ mutants: Pγ 23Bpa, Pγ 30C-MBP, Pγ 50C-MBP, Pγ 73C-MBP and Pγ 84C-MBP. The same region on the gel from a control sample not exposed to UV irradiation was processed for comparison. The number of unique peptide spectra (gray) and the number of total peptide spectra (black) for the α- and β-subunit were determined to calculate the Pβ/Pα ratio. Statistical significance was evaluated by ANOVA analysis using Tukey’s test.
Figure 5
Figure 5
Interaction surfaces of Pγ with the PDE6 catalytic dimer. The refined model for the PDE6 catalytic dimer (α-subunit, blue; β-subunit, light brown) was used to identify Pγ interacting sites on Pαβ. The front (left) and back (right) sides of Pαβ heterodimer are shown. Residues cross-linked with Pγ are represented as magenta spheres.

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