The Molecular Basis for Different Recognition of Substrates by Phosphodiesterase Families 4 and 10

Abstract

SummaryPhosphodiesterases (PDEs) are key enzymes that control the cellular concentrations of the second messengers cAMP and cGMP. The mechanism for selective recognition of substrates cAMP and cGMP by individual PDE families remains a puzzle. To understand the mechanism for substrate recognition by PDE enzymes, the crystal structure of the catalytic domain of an inactive D201N mutant of PDE4D2 in complex with substrate cAMP has been determined at 1.56 Å resolution. The structure shows that Gln369 forms only one hydrogen bond with the adenine of cAMP. This finding provides experimental evidence against the hypothesis of two hydrogen bonds between the invariant glutamine and the substrate cAMP in PDE4, and thus suggests that the widely circulated "glutamine switch" model is unlikely the mechanism for substrate recognition by PDEs. A structure comparison between PDE4D2-cAMP and PDE10A2-cAMP reveals an anti configuration of cAMP in PDE4D2 but syn in PDE10A2, in addition to different contact patterns of cAMP in these two structures. These observations imply that individual PDE families have their characteristic mechanisms for substrate recognition. KeywordsPDE4; cAMP/cGMP; substrate specificity Cyclic nucleotide phosphodiesterases (PDEs) regulate the cellular concentrations of the second messengers cAMP and cGMP and play important roles in many biological processes (1-7). The human genome encodes twenty-one PDE genes that are categorized into eleven families. Alternative mRNA splicing of the PDE genes produces about one hundred proteins (1,3). Selective inhibitors against individual PDE families have been widely studied as therapeutics for treatment of various human diseases (8-15). For example, the PDE5 selective inhibitor sildenafil has been approved for treatment of male erectile dysfunction and pulmonary hypertension (16,17).Individual PDE families have different specificities in hydrolysis of substrates cAMP and cGMP. Thus, PDE families of 4, 7, and 8 prefer to hydrolyze cAMP while PDE5, 6, and 9 are * Correspondence should be addressed to Hengming Ke, Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, NC 27599-7260, USA, Tel: +1-919-966-2244; Fax: +1-919-966-2852; email: hke@med.unc.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Accession codesThe coordinates and structural factors have been deposited into the Protein Data Bank with accession code of 2PW3. cGMP specific. The remaining PDE families 1, 2, 3, 10, and 11 utilize both cAMP and cGMP as substrates, but have slightly different catalytic efficacies. Understanding subst...

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…Catalytic rates in those mutants are like those of WT enzymes. In the x-ray co-crystal of PDE4D2 in complex with cAMP, there is only one hydrogen bond between the glutamine side chain and cAMP compared with the prediction of two bonds (401).…”

“…This seems counterintuitive since 5=-nucleotides interact weakly with PDEs (K i ϳ1-10 mM) compared with the affinities for substrates (K m ϳ0.02-40 M) (399). Wang and co-workers (399,401) have determined the x-ray crystal structures of PDE4D2 (a cAMP-specific PDE) and PDE10 (dual-specificity PDE) utilizing C domains with WT sequence and those in which mutations of critical residues have rendered them largely inactive. The mutations introduced into PDE4D2 or PDE10A2 did not significantly alter the active site conformation compared with WT proteins, thereby validating insights regarding interaction of substrates with WT PDEs.…”

Small Molecule Allosteric Modulators of Phosphodiesterase 4

“…The three inhibitors have a central planar ring that stacks between Phe372 and Ile336, and each forms a hydrogen bond with Gln535. These features are common to all known PDE4 inhibitors (Wang et al 2007b). Active site-directed, competitive PDE4 inhibitors often coordinate to the catalytic metals in the active site.…”

“…The understanding of the active site pocket has allowed the design of inhibitors competitive with cyclic nucleotide binding, and there are sufficient differences between different superfamily members to create family specific inhibitors (e.g., PDE4 vs. PDE5). However, the active site residues of PDE4A, 4B, 4C, and 4D are completely superimposable, and there are no sequence or structural differences that can allow the design of subtype-selective PDE4 competitive inhibitors, for example, PDE4B vs. PDE4D (Wang et al 2007b).…”

“…These are compounds with complex kinetics of inhibition with rolipram being the archetype (Huston et al 1996). The bulk of industry effort has been devoted to the design of active sitedirected PDE4 inhibitors with simple Michaelis-Menten kinetics that bind to the catalytic site and thus are competitive with the cAMP substrate (Wang et al 2007b). Atypical PDE4 inhibitors such as rolipram were recognized to bind both high-and low-affinity sites on PDE4, although the structural basis of those sites was not known (Souness and Rao 1997).…”