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Jacobitz implicated this region by truncation analysis of PDE4A to UCR2 residues
265-332 (Jacobitz et al.
1996
), Fig.
1
, while Rocque and colleagues mapped a
requirement for high-affinity rolipram binding to a similar region within the UCR2
of PDE4B2, namely residues 81-152 (Rocque et al.
1997
). Both groups recognized
that high-affinity rolipram binding required the presence of the catalytic domain
and that further truncation of the enzyme to remove UCR2 yielded proteins with
low-affinity rolipram binding to the catalytic site. Two additional atypical PDE4
inhibitors, RS25344 and PMNPQ, were described by Saldou, who also showed by
truncation analysis a requirement for UCR2 to achieve potent enzyme inhibition
(Saldou et al.
1998
; Brideau et al.
1999
). Whether the high-affinity rolipram binding
site was an allosteric site or instead altered the conformation of the catalytic site to
induce a change from low- to high-affinity rolipram binding was unclear (Jacobitz
et al.
1996
; Houslay
2001
; Liu et al.
2001
; Houslay et al.
2005
).
We initially attempted cocrystallization of a truncated form of PDE4 containing
the catalytic domain and C-terminal sequences. Crystal structures obtained with
(R/S)-rolipram (PDB ID: 3G4K), RS25344 (PDB ID: 3G4I), and PMNPQ (PDB ID:
3G58) revealed a novel binding mode in which chemical elements determining
potency were oriented out of the active site toward solvent. 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 atypical PDE4
inhibitors, in contrast, did not contact the metals, but instead had two aromatic
groups that reached out of the active site and interacted with the solvent (Fig.
2
).
This result was surprising since previous studies had demonstrated that modifica-
tions of either aromatic group could significantly alter the ability to inhibit long
forms of PDE4 (Wilhelm and Axt
1995
). Close examination of the catalytic domain
also showed a groove across the active site that could, potentially, “dock” with a
regulatory domain such as UCR2.
C
N
N
C
C
C
N
PDE4D
C-Terminus
PDE4D
UCR2
PDE4B
UCR2
Fig. 2 Surface rendered view of PDE4 regulatory helices capping the active site. The surface
rendered catalytic domain is shown in
blue
, the C-terminal helix is shown in
yellow
, and UCR2 in
green
. The entire supershort UCR2 was visible in the PDE4B structure, while only the UCR2
gating helix is visible in the PDE4D structure (Burgin et al.
2010
)
