Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies.
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.
Schalk-Hihi
Céline
C
Structural Biology, Johnson & Johnson Pharmaceuticals Research and Development, LLC, Exton, Pennsylvania 19341, USA. cschalkh@prdus.jnj.com
Ma
Hong-Chang
HC
Struble
Geoffrey T
GT
Bayoumy
Shariff
S
Williams
Robyn
R
Devine
Eric
E
Petrounia
Ioanna P
IP
Mezzasalma
Tara
T
Zeng
Lee
L
Schubert
Carsten
C
Grasberger
Bruce
B
Springer
Barry A
BA
Deckman
Ingrid C
IC
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
D050506:Mutant Chimeric Proteins; D047428:Protein Kinase Inhibitors; D020794:Receptor Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor