title: Negative constraints underlie the ErbB specificity of epidermal growth factor-like ligands.
abstract: Epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner, but the molecular basis for this specificity is poorly understood. We have previously shown that certain residues in human EGF (Ser(2)-Asp(3)) and TGFalpha (Glu(26)) are not essential for their binding to ErbB1 but prevent binding to ErbB3 and ErbB4. In the present study, we have used a phage display approach to affinity-optimize the C-terminal linear region of EGF-like growth factors for binding to each ErbB receptor and thereby shown that Arg(45) in EGF impairs binding to both ErbB3 and ErbB4. By omitting all these so-called negative constraints from EGF, we designed a ligand designated panerbin that binds ErbB1, ErbB3, and ErbB4 with similarly high affinity as their wild-type ligands. Homology models, based on the known crystal structure of TGFalpha-bound ErbB1, showed that panerbin is able to bind ErbB1, ErbB3, and ErbB4 in a highly similar manner with respect to position and number of interaction sites. Upon in silico introduction of the experimentally known negative constraints into panerbin, we found that Arg(45) induced local charge repulsion and Glu(26) induced steric hindrance in a receptor-specific manner, whereas Ser(2)-Asp(3) impaired binding due to a disordered conformation. Furthermore, radiolabeled panerbin was used to quantify the level of all three receptors on human breast cancer cells in a single radioreceptor assay. It is concluded that the ErbB specificity of EGF-like growth factors primarily results from the presence of a limited number of residues that impair the unintended interaction with other ErbB receptors.
authors.affiliation: Department of Cell Biology and Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen, Faculty of Science, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. b.vanderwoning@science.ru.nl
authors.forename: Sebastian P
authors.initials: SP
authors.lastname: van der Woning
authors.forename: Walter
authors.initials: W
authors.lastname: van Rotterdam
authors.forename: Sander B
authors.initials: SB
authors.lastname: Nabuurs
authors.forename: Hanka
authors.initials: H
authors.lastname: Venselaar
authors.forename: Saskia
authors.initials: S
authors.lastname: Jacobs-Oomen
authors.forename: Miriam
authors.initials: M
authors.lastname: Wingens
authors.forename: Gert
authors.initials: G
authors.lastname: Vriend
authors.forename: Catelijne
authors.initials: C
authors.lastname: Stortelers
authors.forename: Everardus J J
authors.initials: EJ
authors.lastname: van Zoelen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018739:Oncogene Proteins v-erbB / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D020893:Receptor, ErbB-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066247:Receptor, ErbB-4; D017386:Sequence Homology, Amino Acid
chemical_list: D008024:Ligands; D018739:Oncogene Proteins v-erbB; D004815:Epidermal Growth Factor; C587689:ERBB4 protein, human; D066246:ErbB Receptors; C587690:Erbb4 protein, mouse; D020893:Receptor, ErbB-3; D066247:Receptor, ErbB-4