Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1
ABSTRACT Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.
- SourceAvailable from: Jean-Pierre Changeux
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- "The structurally resolved allosteric KIs known thus far include compounds that inhibit mitogen-activated protein kinase kinase, protein kinase B (AKT), or insulinlike growth factor 1 receptor by occupying a pocket adjacent to the ATP binding site ( " allosteric back-pocket " ) (Ohren et al., 2004; Barnett et al., 2005; Lindsley et al., 2005) or bind to more remote sites, like the myristate binding site (Adrian et al., 2006; Zhang et al., 2009, 2010), the rapamycin binding site of mTOR (Choi et al., 1996; Wang and Sun, 2009; Yang et al., 2013), or the peptide binding site recently discovered in checkpoint kinase 1 (Converso et al., 2009). In addition, targeting the allosteric sites on protein kinases may provide means to identify activators rather than inhibitors that could be useful for therapeutic intervention (Grimsby et al., 2003; Guertin and Grimsby, 2006; Sanders et al., 2007; Hindie et al., 2009). 4. Extracellular Allosteric Modulation of Receptor Tyrosine Kinases. "
ABSTRACT: Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.Pharmacological reviews 10/2014; 66(4):918-47. DOI:10.1124/pr.114.008862 · 18.55 Impact Factor
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- "However, PINK1 contains no known small-molecule binding sites. Another potential strategy might involve manipulation of protein interaction sites or the active site, given that synthetic ligands have been identified that bind to the protein docking sites on the kinase PDK1 (Hindie et al., 2009; Wei et al., 2010) and, separately, that Src activity can be controlled by chemical complementation of an active site catalytic residue, allowing ATP to be accepted only when imidazole was provided to mutant Src (Ferrando et al., 2012; Qiao et al., 2006). However, these approaches were not applicable to PINK1 as no structural data for PINK1 are available. "
ABSTRACT: Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.Cell 08/2013; 154(4):737-47. DOI:10.1016/j.cell.2013.07.030 · 33.12 Impact Factor
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- "PDK1 kinase is a key regulator of AGC kinases, which play crucial roles in physiological processes relevant to metabolism, growth, proliferation and survival . This protein is regulated allosterically by the binding of a phosphopeptide which Biondi and coworkers managed to mimic with a low-molecular-weight activator  and further solved the structure of the complex . As shown in Figure 4, the largest pocket (PKT1) matches the binding site of ATP. "
ABSTRACT: Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse-grained methodology is able to capture the effects triggered by allosteric ligands already described in the literature. Conclusions We introduce a simple computational approach to predict the presence and position of allosteric sites in a protein based on the analysis of changes in protein normal modes upon the binding of a coarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newly curated non-redundant set of 91 proteins with reported allosteric properties. The software developed in this work is available upon request from the authors.BMC Bioinformatics 10/2012; 13(1):273. DOI:10.1186/1471-2105-13-273 · 2.67 Impact Factor