Effect of Zinc and Temperature on the Conformation of the γ Subunit of Retinal Phosphodiesterase:  A Natively Unfolded Protein

Uversky, Vladimir N.; Permyakov, Sergey E.; Zagranichny, Vasily E.; Rodionov, I. L.; Fink, Anthony L.; Cherskaya, Alexandra M.; Wasserman, L. A.; Permyakov, Eugene A.

doi:10.1021/pr0155127

Cited by 65 publications

(59 citation statements)

References 67 publications

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“…Amino acid analysis and biochemical studies of PDE␥ had led to the conclusion that PDE␥ is an IDP (7,8). We found that the peaks in the 2D [ 1 H-15 N]-HSQC NMR spectrum of PDE␥ exhibited narrow linewidths with poorly dispersed amide proton chemical shifts [see supporting information (SI) Fig.…”

Section: Pde␥ Is Intrinsically Disordered But Exhibits Transient Secomentioning

confidence: 99%

“…Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of an IDP, such as PDE␥, confers an advantage in its ability to interact with multiple partners (9).…”

mentioning

confidence: 99%

“…It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of an IDP, such as PDE␥, confers an advantage in its ability to interact with multiple partners (9).…”

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confidence: 99%

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Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Song

Guo

Muradov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

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The retinal phosphodiesterase (PDE6) inhibitory ␥-subunit (PDE␥) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic ␣␤-subunits of PDE6 and the ␣-subunit of transducin (␣t). Detailed structural analysis of PDE␥ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDE␥, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDE␥46-87 when bound to ␣t in the transitionstate complex. NMR mapping of the interaction between PDE␥46-87 and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74 -87 of PDE␥ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to ␣t, is left free at this stage. These results indicate that the interaction between PDE␥ and ␣t during the phototransduction cascade involves the selection of preconfigured transient conformations.NMR spectroscopy ͉ protein recognition ͉ transient structure ͉ visual transduction P hototransduction, the primary event of vision is critically regulated by the ␥-subunit of cGMP phosphodiesterase (PDE␥). In the dark, by binding tightly to the catalytic ␣␤-subunits of cGMP PDE (PDE6) PDE␥ keeps PDE6 inactive; yet upon photoexcitation, interaction of PDE␥ with the ␣-subunit of activated transducin (␣ t ) relieves its inhibitory constraint on PDE6. For termination of phototransduction, PDE␥ interacts with both ␣ t and RGS9, a regulator of G protein signaling, to accelerate ␣ t GTPase activity (1). Beyond phototransduction, the importance of PDE␥ in photoreceptor cell viability is demonstrated by rapid retinal degeneration in the PDE␥-knockout mouse (2), and growing evidence suggests that PDE␥ also interacts with some signaling proteins in nonphototransduction pathways (3).Biochemical studies have shown that the central polycationic region (residues 24-45) and the C-terminal region of PDE␥ constitute two distinct sites of interactions with ␣ t or PDE6 (4). The crystal structure of a ternary complex representing a partial model for the GTPase-activating protein (GAP) complex, consisting of a fragment of PDE␥ (residues 46-87), ␣ t chimera (␣ t/i1 ), and the catalytic core of RGS9, revealed that the C-terminal region of PDE␥ contains three discontinuous helices (5). PDE␥ 46-87 interacts with ␣ t in a GTP-dependent manner, mainly through residues in these helices or their linkers, and the indole side chain of W70 plays the key role in this interaction. Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of...

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Section: Pde␥ Is Intrinsically Disordered But Exhibits Transient Secomentioning

confidence: 99%

mentioning

confidence: 99%

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Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Song

Guo

Muradov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

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“…What has not been elucidated is whether this increased level of folding is critical for nanosphere formation and whether it is a consequence of assembly or a consequence of other parameters, such as temperature and pH. Several studies have previously shown that IDPs can undergo significant conformational alteration upon dimerization and subsequent oligomerization (18,19).…”

mentioning

confidence: 99%

Dissecting Amelogenin Protein Nanospheres

Bromley

Kiss

Lokappa

et al. 2011

Journal of Biological Chemistry

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Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.

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“…The C-terminal region (broadly defined as amino acids 62-87) interacts with the catalytic domain and blocks the catalytic activity (8,9,(13)(14)(15)(16)(17). Structural studies of P␥ in solution indicate that this protein is overall an intrinsically disordered protein (18) but contains ␣-helical secondary structure (␣1, ␣2, and ␣3; Fig. 1A) in its C-terminal region (19); the ␣-helical content within the C-terminal region of P␥ is also observed when this region of P␥ forms a complex with either transducin (20) or a chimeric PDE5/6 catalytic domain (21).…”

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confidence: 99%

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

Zhang

Skiba²,

Cote³

2010

Journal of Biological Chemistry

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The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), 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 P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ 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 ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ 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 P␥ 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 P␥ to effectively relieve its inhibitory constraint. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6)2 is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Transducin binds GTP and releases its activated ␣-subunit (T␣*-GTP) to activate membrane-associated rod PDE6 by relieving the inhibition of the ␥-subunit at the active sites. The activation of PDE6 results in rapid lowering of cGMP levels, closure of cGMP gated ion channels, and hyperpolarization of the cell membrane (1-5).The PDE6 holoenzyme consists of a catalytic dimer of ␣-and ␤-subunits (P␣␤) and two inhibitory ␥-subunits (P␥) that are tightly bound to P␣␤. Considering its small size, the P␥ subunit of rod and cone PDE6 serves a remarkable number of functions (reviewed in Refs. (6 and 7): 1) a primary function of the P␥ subunit is to inhibit catalysis of cGMP by binding to the catalytic domain of PDE6; 2) the P␥ subunit also enhances the binding affinity of cGMP to the regulatory GAF (cGMP-regulated PDEs, certain adenylate cyclases, and the transcription factor Fh1A of bacteria) domain; 3) activated transducin binds to the P␥ subunit, leading to deinhibition of PDE6 at its active site; and 4) during deactivation, the ␥-subunit participates in a protein complex with the RGS9 and o...

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Effect of Zinc and Temperature on the Conformation of the γ Subunit of Retinal Phosphodiesterase: A Natively Unfolded Protein

Cited by 65 publications

References 67 publications

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Dissecting Amelogenin Protein Nanospheres

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

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