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

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 the 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 a differential binding of the γ-subunit to the GAFa domains of the α- and β-subunit are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.

Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

“…The results reveal added mass of the correct size and shape for an Fab fragment at the "top" of the structure, i.e. near the GAFa domain, for the N-terminal epitope tag, and near the catalytic domain for the C-terminal tag, consistent with previous cross-linking results (20,27,64). The distance between the two HA-Fab binding sites is ϳ90 Å (Fig.…”

“…6. These results are contrary to previously published PDE6 GAF domain orientations based on a low resolution map (14), but in good agreement with a recently published model of PDE6 (64). The location of the non-catalytic cGMP binding site, inferred from the fitting of the cone PDE6C GAFa/cGMP crystal structure (9) (PDB ID 3DBA), is shown in Fig.…”

“…6. Although this striking difference in arrangements is surprising, given the nearly identical folds of the PDE6 and PDE2 catalytic domains, it is supported by a recently published comprehensive model of PDE6 constrained by cross-linking and mass spectrometry (64).…”

“…Such interdomain regulation by the 9.7-kDa PDE6␥ polypeptide requires either proximity of GAFa and catalytic domains in the quaternary structure, a highly extended conformation for PDE6␥, or long range allosteric control. Cross-linking studies (20,27,64) support a PDE6␥ extended conformation, with contact made between PDE6␥ and all three domains of the catalytic subunits, but direct structural evidence is lacking.…”

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

“…For example, it was demonstrated that a more integrated view of the subunit arrangement of an intact protein complex can be obtained by examining a sample with XL-MS, native MS, and ion-mobility spectrometry coupled to MS so that the complementary structural MS methods provide additional and orthogonal restraints compared with using XL-MS alone [48]. Advanced computational pipelines can support integration of the different types of data from crosslinking and from other sources -such as complementary experimental or computational methods -to facilitate the unbiased use of crosslinking and other restraints to generate reliable models for protein complexes [49][50][51][52]. This led to the generation of a generic hybrid approach for the accurate modeling of large protein assemblies, where XL-MS together with partial crystal structures of subunits were successfully integrated with modeling.…”