Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution

Abstract

Small-angle X-ray scattering (SAXS), which determines the radius of gyration, R(g), and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca(2+) in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca(2+)-saturated CaM molecule induces structural changes from a 'dumb-bell' shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca(2+)-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca(2+)-saturated CaM molecule. Even in the absence of Ca(2+), the conformational changes of CaM occur on TFP binding, giving a slightly smaller R(g) than Ca(2+)-free CaM alone.

Binding of trifluoperazine to apocalmodulin revealed by a combination of small-angle X-ray scattering and nuclear magnetic resonance

“…5c) (Schumacher et al, 2004). These present results correspond to the previous analysis of the pair distance distribution; uncomplexed apoCaM adopts a dumbbell shape and the 1:5 apoCaM-TFP complex adopts an ellipsoid shape (Matsushima et al, 2000). The Kratky plots and the R g values indicate that 5.0 added equivalents of TFP induce a complete compact globular shape of apoCaM.…”

“…This changes the overall conformation of the protein from the extended structure of apoCaM to the more extended characteristic dumbbell shape of Ca 2+ CaM. This increase in radius of gyration (R g ) of the protein was observed in small-angle X-ray scattering (SAXS) studies (Seaton et al, 1985;Heidorn et al, 1989;Matsushima et al, 1989Matsushima et al, , 2000.…”

“…SAXS studies of Ca 2+ CaM complexed with TFP and W-7 found that the binding of four TFP or two W-7 molecules to one Ca 2+ CaM molecule induces structural changes from a dumbbell shape to a compact globular shape (Osawa et al, 1999;Matsushima et al, 2000). Moreover, conformational changes of apoCaM occur in the presence of five TFP molecules per protein molecule (Matsushima et al, 2000). What number of TFP molecules is required to induce the structural changes of apoCaM?…”

The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non–small cell lung carcinoma cells to DNA double-strand break–induced cell death

“…Phenothiazines are well-characterized calmodulin antagonists (26,27), and this led Chafouleas et al (2) to propose that TFP might augment bleomycin cytotoxicity by inhibiting a putative calmodulin-regulated DNA repair pathway. Recent studies have shown that inositol phosphates serve as cofactors for DNA-PK cs during NHEJ (28 -30).…”

Calmodulin in Complex with Proteins and Small Molecule Ligands: Operating with the Element of Surprise; Implications for Structure-Based Drug Design

“…Both hydrophobic (Val55, Il63, Met71, Met109, Leu116, Met124, Met145) and acidic (Glu11, Glu54, Glu114, Glu120) residues of CaM participate in the complexation. The tertiary structure of CaM changes from an elongated dumb-bell, with exposed hydrophobic surfaces, to a compact globular form upon TFP ligation which hinders its interaction with target proteins [14,82]. In order to clarify the strength of interaction between CaM and TFP in solution, Yamaotsu et al [83] performed a molecular dynamics simulation of the CaM-TFP complex in aqueous solution starting from the crystal structure of the 1:4 complex.…”