Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Abstract

We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long ␣-helices. The subunits cross at the GAF-B/ catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.cGMP activation ͉ GAF domains ͉ PDE-2A T he cyclic nucleotides cAMP and cGMP are ubiquitous intracellular signaling molecules that mediate a vast array of biological processes throughout the body. The means by which these two molecules participate in diverse functions, in different cell types and within single cells, involves tight regulation of the spatial and temporal residence of their concentrations. The phosphodiesterases (PDEs) are a superfamily of enzymes that metabolically inactivate cAMP and cGMP (1) to play key roles in both these aspects of regulation. The PDEs are modular enzymes characterized by a relatively conserved C-terminal catalytic domain and a more variable N-terminal domain involved in regulation of activity, subcellular localization, and interactions with other proteins. There are 11 PDE gene families, with different families encoded by one to four genes, and further diversity derived from alternative splicing. The PDE families differ broadly in specificity and affinity for cAMP and cGMP. Much has been learned about the molecular bases for these differences from studies of X-ray crystal structures of the catalytic domains of

A Phosphodiesterase 2A Isoform Localized to Mitochondria Regulates Respiration

“…During import, PDE2A2 might be proteolytically processed, as suggested by a putative processing site RRG-QQ and the resulting N-terminal Gln, which is the third most abundant N-terminal residue resulting from mitochondrial processing (44). Consistent with the N terminus being more flexible, as typically observed for localization sequences, the PDE2A fragment whose structure was recently solved contained the whole protein except for the N terminus (54).…”

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

“…Of immediate relevance is the structural determination of a PDE5/6 chimeric catalytic domain complexed with P␥70 -87 (21) that shows that P␥ residues 71 and 73 interacting with the PDE5/6 H-loop and its adjacent ␣12 helix within the catalytic domain. Intriguingly, this H-loop (found in all PDE family members whose crystal structures have been determined) is believed to be a major structural element responsible for allosteric regulation of the cGMP-stimulated PDE2 enzyme (46). Although the relevance of the PDE5/6 chimera and PDE2 structures to the structure and regulation of photoreceptor PDE6 remains to be determined, our biochemical study supports the idea that P␥ can interact with the H-loop to modulate catalytic activity distinct from the blocking action of the extreme C terminus of P␥.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…The dimerization contacts of PDEs involve the NH 2 -terminal region (PDEs 2, 4, 5, 6, and 10) and/or contacts in the C domains (PDEs 2, 3, 4, 8 and 11) (183,184,287,427). In some PDEs, dimerization provides regulatory mechanisms such as phosphorylation, ligand binding, and autoinhibition.…”

“…In some cases, their kinetic char-acteristics are indistinguishable from those of the holoenzyme, whereas in other instances there are differences (40,103,297,298). Based on the x-ray crystal structure for a near full-length PDE2, Pandit et al (287) have proposed that autoinhibition is provided by physical constraints imposed by dimer contacts in the C domains and that activation occurs by disruption of that interface (Fig. 3); this is discussed in greater detail in section IIIA.…”