Allosteric regulation and inhibition of protein kinases.

The human genome encodes more than 500 different protein kinases: signaling enzymes with tightly regulated activity. Enzymatic activity within the conserved kinase domain is influenced by numerous regulatory inputs including the binding of regulatory domains, substrates, and the effect of post-translational modifications such as autophosphorylation. Integration of these diverse inputs occurs via allosteric sites that relate signals via networks of amino acid residues to the active site and ensures controlled phosphorylation of kinase substrates. Here, we review mechanisms of allosteric regulation of protein kinases and recent advances in the field.

Structural mechanism of a drug-binding process involving a large conformational change of the protein target.

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.

Kinases on Double Duty: A Review of UniProtKB Annotated Bifunctionality within the Kinome.

Phosphorylation facilitates the regulation of all fundamental biological processes, which has triggered extensive research of protein kinases and their roles in human health and disease. In addition to their phosphotransferase activity, certain kinases have evolved to adopt additional catalytic functions, while others have completely lost all catalytic activity. We searched the Universal Protein Resource Knowledgebase (UniProtKB) database for bifunctional protein kinases and focused on kinases that are critical for bacterial and human cellular homeostasis. These kinases engage in diverse functional roles, ranging from environmental sensing and metabolic regulation to immune-host defense and cell cycle control. Herein, we describe their dual catalytic activities and how they contribute to disease pathogenesis.

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations.

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often "cryptic" without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems.

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast.

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.