title: Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: implications to tetramer stability and ligand-binding.
abstract: Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, P.O. Box 369, 13560-970 São Carlos, SP, Brazil.
authors.forename: Daniela B B
authors.initials: DB
authors.lastname: Trivella
authors.forename: Lucas
authors.initials: L
authors.lastname: Bleicher
authors.forename: Leonardo de Castro
authors.initials: Lde C
authors.lastname: Palmieri
authors.forename: Helton José
authors.initials: HJ
authors.lastname: Wiggers
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: Montanari
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
authors.forename: Luís Maurício T R
authors.initials: LM
authors.lastname: Lima
authors.forename: Débora
authors.initials: D
authors.lastname: Foguel
authors.forename: Igor
authors.initials: I
authors.lastname: Polikarpov
mesh_terms: D000495:Allosteric Site; D019943:Amino Acid Substitution; D000682:Amyloid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000686:Amyloidosis / Q000209:etiology / Q000378:metabolism; D018360:Crystallography, X-Ray; D019833:Genistein / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000682:Amyloid; D008024:Ligands; D050505:Mutant Proteins; D011228:Prealbumin; D011994:Recombinant Proteins; C041120:amyloid prealbumin; D019833:Genistein