The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug.
3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.
Horváth
István
I
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina út 29 Budapest, H-1113 Hungary.
Harmat
Veronika
V
Perczel
András
A
Pálfi
Villo
V
Nyitray
László
L
Nagy
Attila
A
Hlavanda
Emma
E
Náray-Szabó
Gábor
G
Ovádi
Judit
J
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014268:Trifluoperazine / Q000737:chemistry / Q000494:pharmacology*; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D014961:X-Ray Diffraction
D002147:Calmodulin; D018492:Dopamine Antagonists; D014268:Trifluoperazine; D014747:Vinblastine; C107832:deacetoxyvinzolidine; D010727:Phosphoric Diester Hydrolases