Diacylglycerol Signaling

30 P.M. Blumberg et al.
3.4 DAG-Lactones as Manipulable Ligands for C1 Domains
The exogenous natural products identified as high affinity ligands for C1 domains
all possess synthetically complex, constrained backbones that maintain the appropriate
orientation of hydrogen-bonding substituents and hydrophobic regions. The
tigliane backbone of the phorbol esters has eight chiral centers; the macrocyclic
ring of bryostatin 1 has 11. The endogenous ligand, DAG, in contrast, has only a
single chiral center but its simplicity is counterbalanced by its relatively low affinity,
reflecting the flexibility of its conformation. Using the DAG structure as a starting
point, the Marquez group sought to constrain the structure in order to eliminate this
flexibility. The guiding concept was that the constraint imposed upon a flexible
ligand when it binds to its binding site is associated with a loss of entropy, which
translates thermodynamically into a less favorable free energy of binding. If the
ligand in the unbound state can already be constrained into the binding conformation,
then this loss of entropy upon binding will not occur and the free energy of
binding will be correspondingly enhanced. Comparison of different approaches for
constraining DAG revealed that the DAG-lactone structure successfully accomplished
this objective (Marquez et al. 1999). Optimization of the pattern of substitution on
the side chains of the DAG-lactone has then provided ligands with binding affinities
approaching those of the phorbol ester (Nacro et al. 2001), and a convenient stereosynthesis
for the single chiral center has been developed (Kang et al. 2004). Such
DAG-lactones have provided powerful probes for approaching a variety of issues
related to ligand – C1 domain interactions (Marquez and Blumberg 2003).
3.5 Nature of the Interactions of Ligands with the C1 Domain
The three-dimensional structures of multiple DAG-responsive C1 domains have
been solved by NMR (Hommel et al. 1994; Xu et al. 1997), and we were able to
determine the crystal structure of the C1b domain of PKC d in complex with phorbol
ester (Zhang et al. 1995). This structure revealed that the C1 domain functions as a
hydrophobic switch. The phorbol ester inserts into a hydrophilic cleft formed by the
pulled apart strands of a b-sheet at the top of the C1 domain. This upper surface of
the C1 domain surrounding the cleft is hydrophobic and the phorbol ester, upon
insertion, completes the hydrophobic surface, favoring the penetration of the C1
domain – phorbol ester complex into the lipid membrane. The C1 domain does
not appreciably change conformation upon phorbol ester binding, eliminating
possible allosteric models for its mechanism of action. Rather, the binding changes
the association preference of the hydrophobic face of the C1 domain. Two different
structural features of the ligand contribute to the hydrophobicity of the ligand – C1
domain complex. While the first feature is the coverage of the hydrophilic cleft of
the C1 domain, which may be similar between ligands, the second is the contribution
made by the hydrophobic side chains projecting from the ligand, for which