Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase

Abstract

Allostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site and affect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins.

Coupled regulation by the juxtamembrane and sterile α motif (SAM) linker is a hallmark of ephrin tyrosine kinase evolution

“…Conserved allosteric communication networks have been observed in many protein kinases that couple distal protein regions and molecular events such as substrate binding, posttranslational modifications, and protein interactions to the active site in ways that impact catalytic activity (25,27,29,(43)(44)(45)(46). We believe that the SAM linker comprises part of an allosteric communication network in Eph kinases that allows the coupling of other important functional regions to the SAM linker and juxtamembrane.…”

Predicting protein targets for drug-like compounds using transcriptomics

“…biotin-PIP3 (20 nM) was measured using alphascreen technology (Perkin-Elmer), a beadbased proximity assay. The displacement of the interaction by Wortmannin was performed as previously described for the catalytic domain of PDK1 (Schulze et al, 2016;Zhang et al, 2014). Briefly, the assays were performed in a final volume of 25 µL in white 384-well microtiter plates (Greiner Bio-One), including the interacting partners Table S7.…”

“…The concentration of binding partners in the assays were chosen so that both inhibitors and enhancers of the interaction could be identified. Controls using Bio-GST were performed to rule out unspecific effects on the biotin-GST alphascreen interaction assay system.PDK1 protein kinase activity assay:The in vitro activity of PDK1 was tested using 100-300 ng purified protein, following the transfer of 32 P from radiolabelled [g 32 P]ATP to the polypeptide substrate T308tide at room temperature (22 °C) in a mix containing 50 mM Tris pH 7.5, 0.05 mg/ml BSA, 0.1% b -mercaptoethanol, 10 mM MgCl2, 100 µM [g 32 P]ATP (5-50 cpm/pmol) and 0.003% Brij, as previously performed (Schulze et al, 2016). Figure S1.…”

Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile

“…Hexokinase 2 (HK2) is an enzyme that catalyzes the first step of glycolysis, ATP citrate lyase (ACLY) is an important enzyme in lipid biosynthesis [88], Pyruvate dehydrogenase kinase 1 (PDK1) is an enzyme that phosphorylates and inactivates pyruvate dehydrogenase, the enzyme responsible for the first step of citric acid cycle-TCA [89]-and pyruvate kinase isozyme 2 (PKM2) catalyzes the last glycolysis reaction [90]. Although we have not identified any significant differences in the expression of ACLY, PDK1 and HK2, we cannot affirm that there is no metabolic changes in T cells treated with MSC-EVs, since the regulation of these enzymes may act principally at the protein level [91][92][93]. However, importantly, we detected a difference in PKM2 expression.…”