title: Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments.
abstract: Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners.
authors.affiliation: CIRPeB, University of Naples Federico II, via Mezzocannone, 16, 80134, Napoli, Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Calvanese
authors.forename: Annamaria
authors.initials: A
authors.lastname: Sandomenico
authors.forename: Andrea
authors.initials: A
authors.lastname: Caporale
authors.forename: Annalia
authors.initials: A
authors.lastname: Focà
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Focà
authors.forename: Gabriella
authors.initials: G
authors.lastname: D'Auria
authors.forename: Lucia
authors.initials: L
authors.lastname: Falcigno
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
mesh_terms: D030201:Activin Receptors, Type I / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D058851:GPI-Linked Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D008279:Magnetic Resonance Imaging; D062105:Molecular Docking Simulation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D055457:Nodal Protein / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D055457:Nodal Protein; D010455:Peptides; C070636:TDGF1 protein, human; C498932:ACVR1B protein, human; C424205:ACVR1C protein, human; D030201:Activin Receptors, Type I
keywords: NMR; SPR, Nodal; TGF-beta; peptides