title: Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis.
abstract: A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.
authors.affiliation: Dept. of Cancer Biology and Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA. christopher_marshall3@merck.com
authors.forename: C Gary
authors.initials: CG
authors.lastname: Marshall
authors.forename: Maricel
authors.initials: M
authors.lastname: Torrent
authors.forename: Olusegun
authors.initials: O
authors.lastname: Williams
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Hamilton
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Buser
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: C470243:KIF11 protein, human; D000255:Adenosine Triphosphate; D016547:Kinesins