Biology Reference
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different cellular microdomains and for isoform-selective regulation of PDE4
activity by accessory proteins (Houslay et al.
2007
). PDE4 plays an important
role in spatial and temporal patterning of signaling within cAMP microdomains
(Cooper
2003
; Terrin et al.
2006
; Willoughby et al.
2006
; Warrier et al.
2007
).
2 Structure of PDE4 Regulatory Domains
The catalytic domains from seven different PDE superfamily family members
(PDE1, PDE3, PDE4, PDE5, PDE7, PDE9, and PDE10) have been crystallized
including all four PDE4 subtypes (4A, 4B, 4C, 4D) (Ke and Wang
2007
). Compari-
son of these crystal structures shows that all catalytic domains have very similar
overall folds, and the major features of the active site are conserved through evolution
for enzymes able to hydrolyze both cAMP and cGMP to the corresponding monopho-
sphate (Card et al.
2004
). These features include two catalytic metals (magnesium
and zinc) coordinated by histidine residues that activate water during catalysis
(M site). In addition, nucleotide-bound structures show a conserved hydrophobic
clamp (P clamp), which positions the planar purine ring of the nucleotide. Selectivity
for binding adenine or guanine is not the result of different residues making
differential contacts to adenine or guanine, but instead due to the conformation of a
single glutamine residue (Zhang et al.
2004
). The carbonyl and amine groups of
the conserved glutamine make specific hydrogen bonds to the adenine or guanine
base depending upon the conformation of the glutamine residue (Q switch). In some
enzymes, the conformation of the glutamine residue is not constrained resulting
in dual-specific enzymes able to hydrolyze both cAMP and cGMP. The understand-
ing of the active site pocket has allowed the design of inhibitors competitive
with cyclic nucleotide binding, and there are sufficient differences between differ-
ent 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
).
We recently solved the structures of PDE4 regulatory domains with bound
inhibitors (Burgin et al.
2010
). Key to this achievement was cocrystallization
with an atypical PDE4 inhibitor (Saldou et al.
1998
). 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 site-
directed 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
). Clues to the basis of high-affinity rolipram binding
could be gleaned from two different studies where a portion of the regulatory UCR2
domain was implicated in the generation of high-affinity binding of rolipram.
