Structural insight into substrate specificity of phosphodiesterase 10

Abstract

Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity.crystal structure ͉ cyclic nucleotides cAMP and cGMP C yclic nucleotide phosphodiesterases (PDEs) are enzymes hydrolyzing the second messengers adenosine and guanosine 3Ј,5Ј-cyclic monophosphates (cAMP and cGMP). The human genome encodes 21 PDE genes that are categorized into 11 families (1, 2). Selective inhibitors against individual PDE families have been developed as therapeutics for treatment of various human diseases (3-8). The best known examples are the PDE5 inhibitors sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) that have been used for treatment of male erectile dysfunction (5). Sildenafil (Revatio) has also been approved for treatment of pulmonary hypertension (9). PDE10 was independently identified by three groups in 1999 and shows a dual activity on hydrolysis of both cAMP and cGMP (10-12). PDE10 is highly expressed in brain striatum (13-16). Reduction of PDE10A mRNA and protein levels in striatum of transgenic mice implies a role of PDE10A in Huntington's disease (17,18). Knockout mice experiments suggest that PDE10A is involved in regulating striatal output, possibly by reducing the sensitivity of medium spiny neurons to glutamatergic excitation (19). The PDE10 inhibitor papaverine is effective in improving executive function deficits associated with schizophrenia, and therefore inhibition of PDE10 may represent an approach to treatment of psychosis (20,21).PDE families contain a variable N-terminal regulatory domain and a conserved C-terminal catalytic domain. Individual PDE families show different substrate preferences. Crystal structures have been reported for the catalytic domains of seven PDE families in the unliganded form or in complex with inhibitors or products: PDE1B, PDE2A, PDE3B, PDE4B/4D, PDE5A, PDE7A, and PDE9A (22-34). However, it remains a puzzle how the conserved catalytic pocket of the PDE famili...

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

“…In the dual-substrate specific PDE10, the substrates are distinguished through different orientations and interactions of cAMP and cGMP (20). One extra hydrogen bond of cAMP with Gln726 of PDE10A2 in comparison to cGMP may contribute to the 70-fold affinity difference, as shown by K M of 56 nM for cAMP and 4.4 μM for cGMP (20).…”

“…First, a hydrogen bond between the invariant glutamine and a scaffolding tyrosine in the structures of dual substrate specific PDE2 and PDE10 (19,20) will block free rotation of the glutamine side chain and thus prohibit gain of two hydrogen bonds with cAMP or cGMP. In fact, the structures of PDE10A2 in complex with both substrates show that the invariant Gln726 forms two hydrogen bonds with cAMP, but one with cGMP (20).…”

“…Finally, cAMP forms one hydrogen bond with Gln369 of PDE4D2, but two with the invariant glutamine in PDE10A2. These differences suggest that individual PDE families possess characteristic recognition mechanisms for substrate specificity.In the dual-substrate specific PDE10, the substrates are distinguished through different orientations and interactions of cAMP and cGMP (20). One extra hydrogen bond of cAMP with Gln726 of PDE10A2 in comparison to cGMP may contribute to the 70-fold affinity difference, as shown by K M of 56 nM for cAMP and 4.4 μM for cGMP (20).…”

“…However, a couple of lines of evidence suggest that the invariant glutamine may not be the key for the substrate recognition. First, the invariant glutamine forms a hydrogen bond with a tyrosine in the crystal structures of dual-substrate specific PDE2 (19) and PDE10 (20) and is thus unlikely to freely switch its conformation for recognition of the different substrates. Second, the recent PDE10-substrate structures show that cAMP and cGMP have the same syn configuration, but different orientations and interactions (20), suggesting that cAMP and cGMP are recognized by the different contact patterns in PDE10.…”

“…First, the invariant glutamine forms a hydrogen bond with a tyrosine in the crystal structures of dual-substrate specific PDE2 (19) and PDE10 (20) and is thus unlikely to freely switch its conformation for recognition of the different substrates. Second, the recent PDE10-substrate structures show that cAMP and cGMP have the same syn configuration, but different orientations and interactions (20), suggesting that cAMP and cGMP are recognized by the different contact patterns in PDE10. Thus, it is of interest to know if cAMP in PDE4 has the same contact pattern and configuration as that in PDE10 and whether PDE4 and PDE10 have a similar mechanism of substrate recognition.…”