BIOINORGANIC CHEMISTRY A Short Course Second Edition

CALCIUM-DEPENDENT MOLECULES 311

bisindole - based anticancer drug molecules, vinblastine and KAR - 2 [3 ′′ - ( β -
chloroethyl) - 2 ′′ ,4 ′′ - dioxo - 3,5 ′′ - spirooxazolidino - 4 - deacetoxyvinblastine] (see
Figure 6.25 ).^83 The X - ray crystallographic structure of the 1 : 2 CaM:TFP
complex has been deposited in the Protein Data Bank as PDB: 1A29.
The X - ray crystallographic structure of the 1 : 2 CaM:TFP complex (PDB:
1A29) shows binding of one TFP molecule in the C - terminal hydrophobic
pocket, with a second TFP in an interdomain site. In the 1 : 1 structure (PDB:
1CTR), the TFP molecule ’ s tricyclic ring is inverted and rotated 180 ° from that
in the 1 : 2 CaM:TFP complex (PDB: 1A29). The second TFP molecule residing
in the interdomain position shifts toward helix G to make a hydrogen bond
with the side chain of glu127 (N 2 TFP 2 – O ε 2 glu127 = 3.21 Å ).^83 The reference
83 researchers also studied the 1 : 2 CaM:TFP complex using near - UV circular
dichroism (CD) spectroscopy. In the intact system, with a concentration of
20 μ M CaM and varying concentrations of TFP, a negative difference peak
characteristic of CaM:TFP complex formation was observed at 263 nm, reach-
ing a saturating value at a concentration of 75 μ M TFP. At this concentration,
a second positive peak appeared at 270 nm, indicating the binding of the
second TFP molecule. In another experiment, calmodulin was cleaved into
N - and C - terminal fragments by limited trypsinolysis (cleaving CaM along the
central linker at residue lys77). CD spectroscopy of the fragmented CaM was
carried out under the same conditions as the previous experiment. The results
showed similar behavior (formation of the negative difference peak saturating
at 75 μ M TFP) but no evidence of a positive difference peak as seen for the
intact CaM. The conclusion from this experiment is that the disruption of the
CaM central linker region modifi es TFP binding and that the C - terminal (and/
or N - terminal) intradomain sites become saturated with TFP independently.
The position of the second TFP molecule at an interdomain site is confi rmed
by the PDB: 1A29 X - ray crystal structure (see Figure 6.26 ).
In Figure 6.26 , the calmodulin molecule is shown in cartoon format. The
TFP molecules and the calcium ion ligands for calcium sites I and II are shown
in stick format. Bond distances for the calcium site I ligands are collected in
Table 6.8.

Figure 6.25 The anticancer drugs (A) vinblastine and (B) KAR - 2.

NH

N

OH

C 2 H 5

H 3 COOC

H 3 CO N

CH 3

N

C 2 H 5

OCOCH 3

HO COOCH 3

H

Vinblastine KAR-2

O

N

NH

N

OH

C 2 H 5

H 3 COOC

H 3 CO N

CH 3

N

C 2 H 5

O C

H

Cl

A B