Dissecting NGF Interactions with TrkA and p75 Receptors by Structural and Functional Studies of an Anti-NGF Neutralizing Antibody Sonia Covaceuszach 1,2 , Alberto Cassetta 3 , Petr V. Konarev 4,5 , Stefania Gonfloni 2,6 , Rainer Rudolph 7 , Dmitri I. Svergun 4,5 , Doriano Lamba 3 and Antonino Cattaneo 2,8 ⁎ The anti-nerve growth factor (NGF) monoclonal antibody αD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of αD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the αD11 Fab fragment was solved at 1.7 Å resolution. A computational docking model of the αD11 Fab–NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X- ray scattering, provided the structural basis for identifying the residues involved in αD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor. © 2008 Elsevier Ltd. All rights reserved. 1 Lay Line Genomics SpA, via di Castel Romano 100, I-00128 Rome, Italy 2 Neuroscience Program, International School for Advanced Studies, Area Science Park, Basovizza, S.S. 14 Km 163.5, I-34012 Trieste, Italy 3 Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area Science Park, Basovizza, S.S. 14 Km 163.5, I-34012 Trieste, Italy 4 European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany 5 Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia 6 Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, I-00133 Rome, Italy 7 Institut für Biotechnologie, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany 8 European Brain Research Institute, Via del Fosso di Fiorano 64, I-00143 Roma, Italy *Corresponding author. European Brain Research Institute, Via del Fosso di Fiorano 64, I-00143 Roma, Italy. E-mail address: a.cattaneo@ebri.it. Abbreviations used: NGF, nerve growth factor; mAb, monoclonal antibody; SAXS, small-angle X-ray scattering; Trk, tropomyosine receptor kinase; mNGF, mouse nerve growth factor; hNGF, human nerve growth factor; SPR, surface plasmon resonance; MM, molecular mass; rNGF, rat nerve growth factor; rBDNF, rat brain-derived neutrotrophic factor; CDR, complementarity-determining region; ASA, accessible surface area. doi:10.1016/j.jmb.2008.06.008 J. Mol. Biol. (2008) 381, 881–896 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.