Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.
Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.
Kontopidis
George
G
Cyclacel Ltd., James Lindsay Place, Dundee DD1 5JJ, Scotland, United Kingdom. gkontopidis@cyclacel.com
Andrews
Martin J I
MJ
McInnes
Campbell
C
Cowan
Angela
A
Powers
Helen
H
Innes
Lorraine
L
Plater
Andy
A
Griffiths
Gary
G
Paterson
Dougie
D
Zheleva
Daniella I
DI
Lane
David P
DP
Green
Stephen
S
Walkinshaw
Malcolm D
MD
Fischer
Peter M
PM
D020816:Amino Acid Motifs; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry; D018360:Crystallography, X-Ray; D019925:Cyclin A / Q000037:antagonists & inhibitors / Q000737:chemistry; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
C493956:CDKN1A protein, human; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008024:Ligands; D010455:Peptides; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2