Functional Chimeras of the Phosphodiesterase 5 and 10 Tandem GAF Domains

Abstract

The tandem GAF domain of hPDE10A uses cAMP as an allosteric ligand (Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J. E. (2006) J. Biol. Chem. 281, 2841-2846). We used a two-pronged approach to study how discrimination of ligand is achieved in human (h)PDE10A and how domain selection in the phosphodiesterase GAF tandems is determined. First, we examined which functional groups of cAMP are responsible for purine ring discrimination. Changes at the C-6 ring position (removal of the amino group; chloride substitution) and at the N-1 ring position reduced stimulation efficacy by 80%, i.e. marking those positions as decisive for nucleotide discrimination. Second, we generated a GAF tandem chimera that consisted of the cGMP-binding GAF-A unit from hPDE5A1, which signals through cGMP in PDE5, and the GAF-B from hPDE10A1, which signals through cAMP in PDE10. Stimulation of the reporter enzyme exclusively was through the GAF-B domain of hPDE10A1 (EC 50 ‫؍‬ 7 M cAMP) as shown by respective point mutations. The PDE5 GAF-A domain in the chimera did not signal, and its function was reduced to a strictly structural role. Signaling was independent of the origin of the N terminus. Generating 10 additional PDE5/10 tandem GAF chimeras surprisingly demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played an unforeseen decisive role in intramolecular signaling. Swapping the linker sections between PDE5 and PDE10 GAF tandem domains abrogated signaling completely pointing to specific domain interactions within GAF tandems, which are not visible in the available crystal structures with bound ligands.

Dynamic Allostery: Linkers Are Not Merely Flexible

“…Of particular interest, generation of additional PDE5/10 chimeras demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played a key role in intramolecular signaling. Swapping linkers between PDE5 and PDE10 GAF tandem domains abolished signaling (Hofbauer et al, 2008).…”

Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms

“…Human PDE5 GAF-AB domain chimeras showed significant activation by cGMP (Bruder et al, 2006). However, constructs containing either GAF-A or GAF-B PDE5 were unable to respond to cGMP stimulation (Hofbauer et al, 2008), suggesting that both GAF-A and GAF-B domains are necessary for cGMP/GAF domain PDE5 signaling.…”