Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis.
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.
Marshall
C Gary
CG
Dept. of Cancer Biology and Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA. christopher_marshall3@merck.com
Torrent
Maricel
M
Williams
Olusegun
O
Hamilton
Kelly A
KA
Buser
Carolyn A
CA
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
C470243:KIF11 protein, human; D000255:Adenosine Triphosphate; D016547:Kinesins