Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy Luisa Calvanese a,f , Annalia Focà b,f , Annamaria Sandomenico a,b , Giuseppina Focà b , Andrea Caporale a , Nunzianna Doti b , Emanuela Iaccarino c , Antonio Leonardi d , Gabriella D’Auria a,b,e , Menotti Ruvo a,b,⇑ , Lucia Falcigno a,b,e,⇑ a CIRPeB, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy b Institute of Biostructures and Bioimaging, National Research Council, IBB-CNR, via Mezzocannone, 16, 80134 Napoli, Italy c DISTABIF, Università degli Studi della Campania ‘‘Lugi Vanvitelli”, via Vivaldi, 43, 80100 Caserta, Italy d Dept. Medicina Molecolare e Biotecnologie Mediche, Università Federico II di Napoli, Naples, Italy e Dept. of Pharmacy, University of Naples Federico II, via Mezzocannone, 16, 80134 Napoli, Italy article info Article history: Received 21 June 2017 Revised 5 October 2017 Accepted 26 October 2017 Available online xxxx Keywords: Binding Group epitope mapping TGF-beta abstract Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-recep- tor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information pre- viously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor. Ó 2017 Elsevier Ltd. All rights reserved. 1. Introduction Nodal, a member of the transforming growth factor beta (TGF- b) superfamily, is a potent morphogen that regulates the early phases of embryonic development, 1–4 and plays an important role in maintaining pluripotency of embryonic stem cells. 5,6 Normally absent in most adult tissues, Nodal is expressed and active in multiple types of human cancer, including melanoma, 7–9 breast cancer, 10 glioblastoma, 11 prostate cancer. 12,13 The correlation between the inhibition of Nodal signaling and the decrease of aggressive cancer cell features has been well established both in vitro and in vivo, as recently reviewed. 14 Therefore, the presence of Nodal in cancer cells and its absence in normal tissues, makes Nodal an ideal biomarker and a potential antitumor target for inhibiting compounds. Mature Nodal shares with other TGF-b ligands the Smad2/3 canonical activation that starts by TGF-b binding to the extra-cellu- lar domains (ECD) of corresponding receptors. 15 Those involved in Nodal activation are the type I activin receptors ALK4/7 and type II ActRIIA/ActRIIB in cooperation with the co-receptors Cripto-1 (FDGF1) or Cryptic (CFC1). Cripto-1 and Cryptic are glycosyl-phos- phatidyl-inositol (GPI)-linked members of the EGF-CFC family that contain an epidermal growth factor motif (EGF) and a cysteine-rich Cripto-1/FLR1/cryptic (CFC) region. Nodal is reported to establish direct interactions with ALK7 and with Cripto proteins, while the interaction with ALK4 is mediated by Cripto. 16 The interactions of Nodal to ALK4 and ALK7 has been recently described. 17,18 Cripto pro- teins bind Nodal by the EGF region while ALK4 interacts with the CFC region, promoting the recruitment of ActRIIA/B, and facilitating the formation of the multimeric receptor signaling complex. 19,20 This binding triggers intracellular phosphorylation of type I receptor by type II receptor that in turn transmits downstream phosphorylation https://doi.org/10.1016/j.bmc.2017.10.036 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved. Abbreviations: ActRIIA, activin type II receptor A; ActRIIB, activin type II receptor B; ALK, activin receptor-like kinase; CFC, Cripto-1/FLR1/cryptic; ECD, extracellular domain; EGF, epidermal growth factor; GEM, Group epitope mapping; mAb, monoclonal antibody; SMAD, Small Mother against Decapentaplegic; STD, Satura- tion Transfer Difference; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TGF-b, transforming growth factor-beta; TIS, tri-isopropylsilane. ⇑ Corresponding authors at: Istituto di Biostrutture e Bioimmagini del CNR, via Mezzocannone, 16, 80134 Napoli, Italy (M. Ruvo). Dipartimento di Farmacia, Università degli Studi di Napoli ‘‘Federico II”, via Mezzocannone 16, 80134 Napoli, Italy (L. Falcigno). E-mail addresses: menotti.ruvo@unina.it (M. Ruvo), lucia.falcigno@unina.it (L. Falcigno). f These authors equally contributed to the work. Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Please cite this article in press as: Calvanese L., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.036