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

Abstract

The retinal phosphodiesterase (PDE6) inhibitory gamma-subunit (PDEgamma) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic alphabeta-subunits of PDE6 and the alpha-subunit of transducin (alpha(t)). Detailed structural analysis of PDEgamma has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEgamma, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEgamma(46-87) when bound to alpha(t) in the transition-state complex. NMR mapping of the interaction between PDEgamma(46-87) and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74-87 of PDEgamma are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to alpha(t), is left free at this stage. These results indicate that the interaction between PDEgamma and alpha(t) during the phototransduction cascade involves the selection of preconfigured transient conformations.

Fig. 1.

Evidence that PDEγ contains regions of transient secondary structure in solution. (a) [¹H-¹⁵N] NOE of A68-PDEγ plotted as a function of residue number. (b) The ¹H^α secondary shifts and combined ¹³C^α and ¹³C^β secondary shifts (20) observed for PDEγ. The locations of the three helices of PDEγ_46–87 in the partial GAP complex are indicated at the top.

Fig. 2.

PRE results for amide protons in spin-labeled PDEγ. Ten Cys-substituted and spin-labeled variants of PDEγ were analyzed. Dashed lines indicate paramagnetic effects expected for a random coil polypeptide (14).

Fig. 3.

Conformational ensembles representing the solution structure of native PDEγ. Shown in stereoview are the two most populated clusters represented by 56 conformers (a) and 30 conformers (b) from the 100 analyzed structures. The five conformers with lowest energy from each cluster are shown; they are colored from blue at the N terminus to red at the C terminus. Residues R24, K45, D52, and E81 are labeled to delineate the boundaries of two oppositely charged segments.

Fig. 4.

Overlaid 2D [¹H-¹⁵N]-HSQC spectra highlight the spectral changes of A68-PDEγ_46–87 upon binding to the PDE5/6 catalytic domain. Assigned peaks from free A68-PDEγ_46–87 are labeled in red, and those for the complex form are labeled in black. The side-chain imino group of W70 and side-chain amide group of Q83 are labeled with the respective residue name followed by a superscript ε.

Fig. 5.

The conformation of PDEγ favorable for binding α_t is populated in solution. (a) The structure of PDEγ (blue ribbon) in complex with α_t/i1 and RGS9 (surface representation) (5). (b) Superposition of the solution structure of native PDEγ with that in the partial GAP complex.