The Molecular Tweezer CLR01 Stabilizes a Disordered Protein–Protein Interface

Protein regions that are involved in protein–protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein–protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular “Janus” ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein–protein interface, “freezes” one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

[1]  István Simon,et al.  The expanding view of protein–protein interactions: complexes involving intrinsically disordered proteins , 2011, Physical biology.

[2]  M. Takeda,et al.  Phosphorylation of tau at Ser214 mediates its interaction with 14‐3‐3 protein: implications for the mechanism of tau aggregation , 2009, Journal of neurochemistry.

[3]  J. Cherfils,et al.  Dual Specificity of the Interfacial Inhibitor Brefeldin A for Arf Proteins and Sec7 Domains* , 2006, Journal of Biological Chemistry.

[4]  Wei Zu Chen,et al.  A fast empirical approach to binding free energy calculations based on protein interface information. , 2002, Protein engineering.

[5]  J. Janin.  A minimal model of protein–protein binding affinities , 2014, Protein science : a publication of the Protein Society.

[6]  M. Brucale,et al.  The chaperone-like protein 14-3-3η interacts with human α-synuclein aggregation intermediates rerouting the amyloidogenic pathway and reducing α-synuclein cellular toxicity. , 2014, Human molecular genetics.

[7]  J. S. Sodhi,et al.  Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. , 2004, Journal of molecular biology.

[8]  D W Banner,et al.  Molecular recognition at the thrombin active site: structure-based design and synthesis of potent and selective thrombin inhibitors and the X-ray crystal structures of two thrombin-inhibitor complexes. , 1997, Chemistry & biology.

[9]  T. Schulz-Gasch,et al.  Structure-assisted discovery of an aminothiazole derivative as a lead molecule for inhibition of bacterial fatty-acid synthesis , 2007, Acta crystallographica. Section D, Biological crystallography.

[10]  H. Dyson,et al.  Linking folding and binding. , 2009, Current opinion in structural biology.

[11]  J. Acker,et al.  Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. , 1999, Molecular cell.

[12]  Gerhard Klebe,et al.  Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution. , 2007, Journal of molecular biology.

[13]  VINCENT ZOETE,et al.  SwissParam: A fast force field generation tool for small organic molecules , 2011, J. Comput. Chem..

[14]  J. Forman-Kay,et al.  Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis , 2012, Molecular biology of the cell.

[15]  D. Beach,et al.  Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: Evidence for multiple roles of mitotic cyclins , 1991, Cell.

[16]  L. M. Stevers,et al.  Modulators of 14-3-3 Protein–Protein Interactions , 2017, Journal of medicinal chemistry.

[17]  F. Diederich,et al.  Aromatic rings in chemical and biological recognition: energetics and structures. , 2011, Angewandte Chemie.

[18]  C. Ottmann,et al.  A semisynthetic fusicoccane stabilizes a protein-protein interaction and enhances the expression of K+ channels at the cell surface. , 2013, Chemistry & biology.

[19]  Christophe Chipot,et al.  Standard binding free energies from computer simulations: What is the best strategy? , 2013, Journal of chemical theory and computation.

[20]  C. Ottmann,et al.  A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14‐3‐3 , 2017, Angewandte Chemie.

[21]  M. Ultsch,et al.  Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. , 1992, Science.

[22]  J. Forman-Kay,et al.  From sequence and forces to structure, function, and evolution of intrinsically disordered proteins. , 2013, Structure.

[23]  M. Skwarczynska,et al.  Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers. , 2013, ACS chemical biology.

[24]  Markus Kaiser,et al.  Small-molecule stabilization of protein-protein interactions: an underestimated concept in drug discovery? , 2012, Angewandte Chemie.

[25]  J. Lehn,et al.  Janus molecules: Synthesis of double-headed heterocycles containing two identical hydrogen bonding arrays , 1994 .

[26]  Nicholas P. Power,et al.  Protein camouflage in cytochrome c–calixarene complexes , 2012, Nature Chemistry.

[27]  Thomas Schrader,et al.  Molecular tweezer and clip in aqueous solution: unexpected self-assembly, powerful host-guest complex formation, quantum chemical 1H NMR shift calculation. , 2006, Journal of the American Chemical Society.

[28]  G. Bitan,et al.  Molecular tweezers inhibit islet amyloid polypeptide assembly and toxicity by a new mechanism. , 2015, ACS chemical biology.

[29]  A. Iglesias,et al.  Intrinsic disorder is a key characteristic in partners that bind 14‐3‐3 proteins , 2006, Proteins.

[30]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[31]  C. Ochsenfeld,et al.  Molecular tweezers with varying anions: a comparative study. , 2013, The Journal of organic chemistry.

[32]  S. C. Rogers,et al.  QUASI: A general purpose implementation of the QM/MM approach and its application to problems in catalysis , 2003 .

[33]  V. Uversky.  Unusual biophysics of intrinsically disordered proteins. , 2013, Biochimica et biophysica acta.

[34]  Christian Ottmann,et al.  Structure of the p53 C‐terminus bound to 14‐3‐3: Implications for stabilization of the p53 tetramer , 2010, FEBS letters.

[35]  P. Russell,et al.  Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein , 1999, Nature.

[36]  W Smith,et al.  DL_POLY_2.0: a general-purpose parallel molecular dynamics simulation package. , 1996, Journal of molecular graphics.

[37]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[38]  J. Cherfils,et al.  Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor , 2003, Nature.

[39]  T. Schrader,et al.  A molecular tweezer for lysine and arginine. , 2005, Journal of the American Chemical Society.

[40]  A. Fersht,et al.  14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers , 2008, Nucleic acids research.

[41]  C. Ottmann,et al.  Molecular tweezers modulate 14-3-3 protein-protein interactions. , 2013, Nature chemistry.

[42]  H. Dyson,et al.  Intrinsically unstructured proteins and their functions , 2005, Nature Reviews Molecular Cell Biology.

[43]  G. Torrie,et al.  Nonphysical sampling distributions in Monte Carlo free-energy estimation: Umbrella sampling , 1977 .

[44]  C. Ottmann,et al.  Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer. , 2012, Journal of molecular biology.

[45]  J. Daly,et al.  Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Hans W. Horn,et al.  ELECTRONIC STRUCTURE CALCULATIONS ON WORKSTATION COMPUTERS: THE PROGRAM SYSTEM TURBOMOLE , 1989 .

[47]  C. Ottmann,et al.  Stabilization of Protein-Protein Interactions in chemical biology and drug discovery. , 2015, Progress in biophysics and molecular biology.

[48]  T. Kunitake,et al.  Mesoscopic supramolecular assembly of a ‘Janus’ molecule and a melamine derivative via complementary hydrogen bonds , 1995 .

[49]  J. Rudolph,et al.  Cdc25 phosphatases and cancer. , 2004, Chemistry & biology.

[50]  S. Zimmerman.  Rigid Molecular Tweezers as Hosts for the Complexation of Neutral Guests , 1993 .

[51]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[52]  S. Zimmerman.  A journey in bioinspired supramolecular chemistry: from molecular tweezers to small molecules that target myotonic dystrophy , 2016, Beilstein journal of organic chemistry.

[53]  J. Forman-Kay,et al.  Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions , 2013, Proceedings of the National Academy of Sciences.

[54]  R. Sunahara,et al.  Interaction of Gsα with the Cytosolic Domains of Mammalian Adenylyl Cyclase* , 1997, The Journal of Biological Chemistry.

[55]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[56]  C. Ottmann,et al.  Identification and structural characterization of two 14-3-3 binding sites in the human peptidylarginine deiminase type VI. , 2012, Journal of structural biology.

[57]  T. Schrader,et al.  Aromatic interactions by molecular tweezers and clips in chemical and biological systems. , 2013, Accounts of chemical research.

[58]  Christophe Chipot,et al.  Frontiers in free‐energy calculations of biological systems , 2014 .

[59]  S R Sprang,et al.  Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS. , 1997 .

[60]  C. Peng,et al.  Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. , 1997, Science.

[61]  L. M. Stevers,et al.  Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR , 2016, Proceedings of the National Academy of Sciences.

[62]  C. Ottmann,et al.  Modulators of protein-protein interactions. , 2014, Chemical reviews.

[63]  C. Ottmann,et al.  Structure of a 14-3-3 coordinated hexamer of the plant plasma membrane H+ -ATPase by combining X-ray crystallography and electron cryomicroscopy. , 2007, Molecular cell.

[64]  R. Swendsen,et al.  THE weighted histogram analysis method for free‐energy calculations on biomolecules. I. The method , 1992 .

[65]  C. Ottmann,et al.  Structural insights of the MLF1/14‐3‐3 interaction , 2012, The FEBS journal.

[66]  Fabrizio Giordanetto,et al.  Stabilization of protein-protein interactions by small molecules. , 2014, Drug discovery today.

[67]  J. Wells,et al.  Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. , 2014, Chemistry & biology.

[68]  Michael Hann,et al.  Stabilization of protein-protein interactions in drug discovery , 2017, Expert opinion on drug discovery.

[69]  C. Ottmann,et al.  Involvement of 14‐3‐3 in tubulin instability and impaired axon development is mediated by Tau , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[70]  S. Noskov,et al.  Free energy decomposition of protein-protein interactions. , 2001, Biophysical journal.

[71]  C. Ottmann,et al.  Supramolecular Chemistry Targeting Proteins , 2017, Journal of the American Chemical Society.

[72]  A Keith Dunker,et al.  Signal transduction via unstructured protein conduits. , 2008, Nature chemical biology.