Competition of Ligands and the 18-mer Binding Domain of the RHAU Helicase for G-Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G-quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G-quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G-quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G-quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu-ttpy (ttpy=4'-tolyl-2,2':6',2''-terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug-target interactions, but also how the binding to other partners is affected.

[1]  D. Loew,et al.  Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes. , 2020, Biochemistry.

[2]  F. Diederich,et al.  Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor , 2018, Chemical science.

[3]  W. S. Hopkins,et al.  What stoichiometries determined by mass spectrometry reveal about the ligand binding mode to G-quadruplex nucleic acids. , 2017, Biochimica et biophysica acta. General subjects.

[4]  V. Birkedal,et al.  A direct view of the complex multi-pathway folding of telomeric G-quadruplexes , 2016, Nucleic acids research.

[5]  Jie Yan,et al.  RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis , 2016, Nucleic acids research.

[6]  Adrien Marchand,et al.  Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G-Quadruplexes. , 2016, Chemistry.

[7]  S. Neidle.  Quadruplex Nucleic Acids as Novel Therapeutic Targets. , 2016, Journal of medicinal chemistry.

[8]  Jean-Louis Mergny,et al.  G-quadruplexes and helicases , 2016, Nucleic acids research.

[9]  V. Birkedal,et al.  Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy , 2015, Nucleic acids research.

[10]  A. Phan,et al.  Insights into G-quadruplex specific recognition by the DEAH-box helicase RHAU: Solution structure of a peptide–quadruplex complex , 2015, Proceedings of the National Academy of Sciences.

[11]  J. Mergny,et al.  A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands , 2015, Nucleic acids research.

[12]  S. Balasubramanian,et al.  Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase , 2015, Nucleic acids research.

[13]  Yue Ma,et al.  Ligand-induced conformational changes with cation ejection upon binding to human telomeric DNA G-quadruplexes. , 2015, Journal of the American Chemical Society.

[14]  Adrien Marchand,et al.  Native Electrospray Mass Spectrometry of DNA G-Quadruplexes in Potassium Solution , 2014, Journal of The American Society for Mass Spectrometry.

[15]  A. Phan,et al.  Solution structure of a G-quadruplex bound to the bisquinolinium compound Phen-DC(3). , 2014, Angewandte Chemie.

[16]  E. Novellino,et al.  Differential scanning calorimetry to investigate G-quadruplexes structural stability. , 2013, Methods.

[17]  Danzhou Yang,et al.  Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes , 2013, Nucleic acids research.

[18]  A. Phan,et al.  Monomer-dimer equilibrium for the 5'-5' stacking of propeller-type parallel-stranded G-quadruplexes: NMR structural study. , 2012, Chemistry.

[19]  A. Phan,et al.  NMR spectroscopy of G-quadruplexes. , 2012, Methods.

[20]  A. Serero,et al.  Formation of pearl-necklace monomorphic G-quadruplexes in the human CEB25 minisatellite. , 2012, Journal of the American Chemical Society.

[21]  E. De Pauw,et al.  Tridentate N-donor palladium(II) complexes as efficient coordinating quadruplex DNA binders. , 2011, Chemistry.

[22]  E. Novellino,et al.  Topological characterization of nucleic acid G-quadruplexes by UV absorption and circular dichroism. , 2011, Angewandte Chemie.

[23]  J. Mergny,et al.  Recognition of G-quadruplex DNA by triangular star-shaped compounds: with or without side chains? , 2011, Chemistry.

[24]  Kah Wai Lim,et al.  Coexistence of two distinct G-quadruplex conformations in the hTERT promoter. , 2010, Journal of the American Chemical Society.

[25]  Mona Singh,et al.  G-Quadruplex DNA Sequences Are Evolutionarily Conserved and Associated with Distinct Genomic Features in Saccharomyces cerevisiae , 2010, PLoS Comput. Biol..

[26]  A. Phan.  Human telomeric G‐quadruplex: structures of DNA and RNA sequences , 2010, The FEBS journal.

[27]  Amy Lin,et al.  Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis. , 2009, Cancer research.

[28]  E. Hovig,et al.  The disruptive positions in human G-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts , 2009, Nucleic acids research.

[29]  B. Pagano,et al.  Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes , 2009, International journal of molecular sciences.

[30]  A. Phan,et al.  Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers. , 2009, Journal of the American Chemical Society.

[31]  E. De Pauw,et al.  Electrospray mass spectrometry to study drug-nucleic acids interactions. , 2008, Biochimie.

[32]  Kwok‐yin Wong,et al.  G‐Quadruplexes: Targets in Anticancer Drug Design , 2008, ChemMedChem.

[33]  M. Teulade‐Fichou,et al.  A hitchhiker's guide to G-quadruplex ligands. , 2008, Organic & biomolecular chemistry.

[34]  J. Redman.  Surface plasmon resonance for probing quadruplex folding and interactions with proteins and small molecules. , 2007, Methods.

[35]  Mateus Webba Da Silva.  NMR methods for studying quadruplex nucleic acids. , 2007 .

[36]  Sarah W. Burge,et al.  Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. , 2007, Journal of the American Chemical Society.

[37]  J. Mergny,et al.  Highly efficient G-quadruplex recognition by bisquinolinium compounds. , 2007, Journal of the American Chemical Society.

[38]  Sarah W. Burge,et al.  Quadruplex DNA: sequence, topology and structure , 2006, Nucleic acids research.

[39]  Dinshaw J. Patel,et al.  Structure of the human telomere in K+ solution: an intramolecular (3 + 1) G-quadruplex scaffold. , 2006, Journal of the American Chemical Society.

[40]  B. Dutrillaux,et al.  Preferential binding of a G-quadruplex ligand to human chromosome ends , 2005, Nucleic acids research.

[41]  P. Borer,et al.  Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. , 2004, Nucleic acids research.

[42]  D. Bearss,et al.  Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Jean-Louis Mergny,et al.  Following G‐quartet formation by UV‐spectroscopy , 1998, FEBS letters.

[44]  P. Kuzmič,et al.  Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. , 1996, Analytical biochemistry.

[45]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[46]  J. Mergny,et al.  Role of Alkali Metal Ions in G-Quadruplex Nucleic Acid Structure and Stability. , 2016, Metal ions in life sciences.

[47]  P. A. Rachwal,et al.  Quadruplex melting. , 2007, Methods.