title: Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.
abstract: This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetramer binding ligand 3 in a 4:4 stoichiometry is also reported. NanoESI-MS-based results match the current understanding of the investigated system and are in agreement with the X-ray structural data, but provide additional mechanistic insight on the ligand binding, due to the better dynamic resolution. This method offers a powerful approach for studying other proteins with allosteric binding sites, as well.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Dragana
authors.initials: D
authors.lastname: Cubrilovic
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Haap
authors.forename: Konstantin
authors.initials: K
authors.lastname: Barylyuk
authors.forename: Armin
authors.initials: A
authors.lastname: Ruf
authors.forename: Martin
authors.initials: M
authors.lastname: Badertscher
authors.forename: Marcel
authors.initials: M
authors.lastname: Gubler
authors.forename: Tim
authors.initials: T
authors.lastname: Tetaz
authors.forename: Catherine
authors.initials: C
authors.lastname: Joseph
authors.forename: Jörg
authors.initials: J
authors.lastname: Benz
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D000494:Allosteric Regulation; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D054852:Small Molecule Libraries; D006597:Fructose-Bisphosphatase