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.

Structural insights on P31‐43, a gliadin peptide able to promote an innate but not an adaptive response in celiac disease

“…Peptide tridimensional models from NMR data were obtained by adopting the standard protocol consisting in the assignment of the proton chemical shifts, integration of the dipolar effects (NOE), conversion of NOE intensities into interproton distances, and calculation of peptide structures compatible with the entire set of distance restraints. NMR spectra were analysed by using the CARA programme (http://www.nmr.ch) as previously reported . In particular, NOE intensities were evaluated by integration of cross‐peaks in 300‐ms ROESY maps and converted into interproton distances, d ij , by the CALIBA programme, by using d HD2‐HD3 of Pro or Pro at 2.2 Å as calibration distance.…”

Expression of Nodal on Bronchial Epithelial Cells Influenced by Lung Microbes Through DNA Methylation Modulates the Differentiation of T-Helper Cells

“…Nodal also further activates its own expression via a positive feedback loop [15]. Nodal signaling is tightly regulated by Cripto, an extracellular GPI-linked protein that acts as a cofactor, while inhibited by Lefty and Cerberus [16]. However, the function of Nodal in the adult human airway epithelia remains unclear.…”

New Anti-Nodal Monoclonal Antibodies Targeting the Nodal Pre-Helix Loop Involved in Cripto-1 Binding

“…Remarkably, 3D1 also blocks in vitro the interaction of Nodal with Cripto-1, a feature of particular relevance in the context of tumor development mechanisms involving the activation of the Smad2/3 axis [ 32 ]. As recently shown [ 20 , 21 ], Nodal also binds to ALK4 and the region of binding seems to be shared with Cripto-1 and ALK7, two well-established Nodal (co)-receptors. On this basis we could speculate that 3D1 has the potential to also inhibit the interaction of Nodal with other receptors thus strengthening inhibition of Smad 2/3 activation and potentiating the anti-tumoral activity of the antibody [ 32 ].…”

“…It is well established that Cripto-1 is essential for the cellular activation of Nodal/ALK4/Smad pathway [ 18 ] and that it may directly interact with ALK4 to enhance the responsiveness of the ALK7/ActRIIB complex to Nodal [ 19 ]. Recently we have reported that Nodal is also capable of physically interacting with both ALK7 and ALK4 regardless of the Cripto-1 presence [ 20 , 21 ]. Although the exact mechanisms underlying the synergistic effect exerted by Cripto-1 on the cellular activation of Nodal-dependent Smad signalling have not been completely understood, the detection of Cripto-1 in melanoma cells [ 5 , 22 , 23 ] supports the relevant role this co-receptor can play in co-promoting tumorigenesis.…”