Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins Diacylglycerol (DAG) is a paradigmatic lipid second messenger in metazoan cell signaling, the first to be discovered. The protein kinase C family is the best known target for DAG signals, but other proteins, such as the chimaerins, have also come to the fore as important receptors. PKCs and other DAG receptors are therapeutic targets for cancer, diabetes, and immunosuppression, among others. PKCs and other DAG receptors bind DAG through their C1 domains. This binding event triggers both membrane translocation and allosteric activation through conformational changes. This project aims to characterize these activation mechanisms at atomic-level detail. In addition to the structure of the phorbol ester:PKC delta C1B domain complex , past work in this project has yielded the first and only structure of a full-length C1 domain protein, that of beta2 chimaerin. The approaches used are 1) the crystallization of full-length C1-domain containing proteins;2) analysis of activation dynamics using molecular simulations;and 3) spectroscopic analysis of activation kinetics and the conformation of active, membrane-associated states inaccessible to crystallization. A major milestone was reached in the last FY. The crystal structure of full-length protein kinase C bII was determined at 4.0 in a partially open conformation. The C1B, C2, and tris-phosphorylated catalytic domains were visualized, while the pseudosubstrate and C1A domains were disordered. The C1B domain clamps the novel helix 624-634 in a position that sequesters the ATP-binding Phe629 of the conserved NFD motif in a catalytically unproductive conformation. A low resolution solution structure of the closed conformation of PKCbII was derived from small angle x-ray scattering. Together, these results show how PKCbII is allosterically regulated by clamping of the NFD helix by the C1B domain. The clamp is reversed upon membrane binding, defining a new structural class of protein kinase regulation.
| Leonard, Thomas A; Ró?ycki, Bartosz; Saidi, Layla F et al. (2011) Crystal structure and allosteric activation of protein kinase C ?II. Cell 144:55-66 |
