title: Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies.
abstract: A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.
authors.affiliation: Structural Biology, Johnson & Johnson Pharmaceuticals Research and Development, LLC, Exton, Pennsylvania 19341, USA. cschalkh@prdus.jnj.com
authors.forename: Céline
authors.initials: C
authors.lastname: Schalk-Hihi
authors.forename: Hong-Chang
authors.initials: HC
authors.lastname: Ma
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Struble
authors.forename: Shariff
authors.initials: S
authors.lastname: Bayoumy
authors.forename: Robyn
authors.initials: R
authors.lastname: Williams
authors.forename: Eric
authors.initials: E
authors.lastname: Devine
authors.forename: Ioanna P
authors.initials: IP
authors.lastname: Petrounia
authors.forename: Tara
authors.initials: T
authors.lastname: Mezzasalma
authors.forename: Lee
authors.initials: L
authors.lastname: Zeng
authors.forename: Carsten
authors.initials: C
authors.lastname: Schubert
authors.forename: Bruce
authors.initials: B
authors.lastname: Grasberger
authors.forename: Barry A
authors.initials: BA
authors.lastname: Springer
authors.forename: Ingrid C
authors.initials: IC
authors.lastname: Deckman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003460:Crystallization; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000138:chemical synthesis / Q000235:genetics; D015202:Protein Engineering; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000138:chemical synthesis* / Q000235:genetics*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D016415:Sequence Alignment; D018411:Spodoptera
chemical_list: D050506:Mutant Chimeric Proteins; D047428:Protein Kinase Inhibitors; D020794:Receptor Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor