Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations which have also been referred to as DFG-out in the literature but which do not have a fully formed allosteric pocket. We have completed a structural analysis of almost two hundred small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that non-classical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two datasets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity.

The metabolic enzyme IMPDH assembles into octamers that can polymerize and form micron-scale structures in cells. Octamers can adopt active, expanded or inactive, compressed conformations driven by allosteric nucleotide and substrate binding. Both forms are accommodated within polymers, and polymerization alone does not alter catalytic activity.

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The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from ∼15°to 38°. An important issue for understanding the structure of M2TM is the role of peptide-lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date-each defined by a different set of criteria and in a different environment-might provide snapshots of the distinct conformational states sampled by the protein.Keywords: M2 proton channel; EPR spectroscopy; site-directed spin labeling; membrane protein structure; peptide-lipid interactions; hydrophobic mismatch; helix tilt; lateral pressure The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. Energetics of formation of M2TM have been studied by using analytical ultracentrifugation (Salom et al. 2000;Howard et al. 2002) and thiol-disulfide equilibria (Cristian et al. 2003a,b). A high-resolution crystal structure has not been solved, although a variety of structural models have been proposed based on site-directed mutagenesis in conjunction with computer modeling (Pinto et al. 1997), molecular dynamics calculations (Zhong et al. 2000), infrared spectroscopy (Torres et al. 2000), and solid-state nuclear magnetic resonance spectroscopy (SSNMR) (Kovacs et al. 2000;Wang et al. 2001;Nishimura et al. 2002). The proposed structures are in good agreement with respect to the identities of the side chains lining the pore, the presence of a water-filled pore near the center of the channel, and the packing of monomers with a left-handed tilt. One detail in which models do differ is in the degree of helix tilt, with proposed tilts ranging from approximately 15°to 38°. The 38°angle structure is based on an abundance of high-resolution SSNMR orientational restraints and a single distance restraint (Nishimura et al. 2002), while the 15°angle structure is based on site-directed mutagenesis, and explains a large body of electrophysiological data for this chan...

Protein and lipid kinases play key regulatory roles in a number of biological processes. Unsurprisingly, activating mutations in kinases have been linked to a number of disorders and diseases, most notably cancers. Thus, kinases have emerged as promising clinical targets. There are more than 500 human protein kinases and about 20 lipid kinases. Most protein kinases share a highly conserved domain, the eukaryotic protein kinase (ePK) domain, which contains the ATP and substrate‐binding sites. Many inhibitors in clinical use bind to the highly conserved ATP binding site. For this reason, many kinase inhibitors are not exclusively selective for their intended targets. Furthermore, despite the current interest in kinase inhibitors, very few kinases implicated in disease have validated inhibitors. This unit describes the human kinome, ePK structure, and types of kinase inhibitors, focusing on methods to identify potent and selective kinase inhibitors. Curr. Protoc. Pharmacol. 60:2.9.1‐2.9.14. © 2013 by John Wiley & Sons, Inc.