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      Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

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          Abstract

          Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo–electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical “active” conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.

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          Most cited references35

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          The structural basis for 14-3-3:phosphopeptide binding specificity.

          The 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3zeta complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 A resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide-protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl.
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            Protein kinases: evolution of dynamic regulatory proteins.

            Eukayotic protein kinases evolved as a family of highly dynamic molecules with strictly organized internal architecture. A single hydrophobic F-helix serves as a central scaffold for assembly of the entire molecule. Two non-consecutive hydrophobic structures termed "spines" anchor all the elements important for catalysis to the F-helix. They make firm, but flexible, connections within the molecule, providing a high level of internal dynamics of the protein kinase. During the course of evolution, protein kinases developed a universal regulatory mechanism associated with a large activation segment that can be dynamically folded and unfolded in the course of cell functioning. Protein kinases thus represent a unique, highly dynamic, and precisely regulated set of switches that control most biological events in eukaryotic cells. Copyright © 2010. Published by Elsevier Ltd.
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              Is Open Access

              Sampling the conformational space of the catalytic subunit of human γ-secretase

              Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain. DOI: http://dx.doi.org/10.7554/eLife.11182.001
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                Author and article information

                Journal
                Science
                Science
                American Association for the Advancement of Science (AAAS)
                0036-8075
                1095-9203
                October 03 2019
                October 04 2019
                October 04 2019
                September 19 2019
                : 366
                : 6461
                : 109-115
                Article
                10.1126/science.aay0543
                7007921
                31604311
                a7c35500-2623-4718-aacf-2e50982b423e
                © 2019

                http://www.sciencemag.org/about/science-licenses-journal-article-reuse

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