Trifluoperazine-induced conformational change in Ca2+-calmodulin

Vandonselaar, M.; Hickie, Robert A.; Quail, J. Wilson; Delbaere, L. T. J.

doi:10.1038/nsb1194-795

Cited by 213 publications

(264 citation statements)

References 37 publications

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“…For example, troponin C binds two TFP molecules (PDB 1WRK) and calmodulin binds TFP with a range of stoichiometries; 1∶1 (PDB 1CTR), 1∶2 (PDB 1A29), and 1∶4 (PDB 1LIN) (28)(29)(30). Even though S100A4, troponin C, and calmodulin are built upon the same basic four-helical structural module, the architectures of the TFP binding pockets, and the positions and orientations of the bound TFP molecules are quite different amongst the three proteins (Fig.…”

Section: Discussionmentioning

confidence: 99%

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Malashkevich

Dulyaninova

Ramagopal

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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S100A4, a member of the S100 family of Ca 2þ -binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensorbased assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 Å crystal structure of human Ca 2þ -S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca 2þ -S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca 2þ -S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca 2þ -S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/ myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.calcium | X-ray crystallography | NMR | small molecule inhibitor | metastasis T he S100 proteins, of which there are more than 20 members, are characterized by their solubility in 100% saturated ammonium sulfate (1 and 2). Each S100 family member contains two Ca 2þ -binding loops; a C-terminal "typical" EF-hand comprised of 12 residues and an N-terminal pseudo EF-hand consisting of 14 residues. The basic organization of the S100 proteins is a symmetric, antiparallel homodimer, in which the N-and C-terminal helices (helices 1 and 4) from each subunit interact to form a stable four helix bundle that serves as the dimer interface. Calcium binding to the C-terminal typical EF-hand significantly alters the angle between helices 3 and 4, which flank the C-terminal Ca 2þ -binding loop, and exposes a hydrophobic cleft that constitutes a binding surface for target proteins (3-5). Thus the S100 proteins operate as calcium-activated switches that bind and regulate the activity of diverse protein targets. S100 proteins are expressed in a tissue and cell specific manner. Elevated expression of individual family members is associated with a number of human pathologies, including cardiomyopathies, cancer, neurodegeneration, and in...

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Section: Discussionmentioning

confidence: 99%

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Malashkevich

Dulyaninova

Ramagopal

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Binding the small-molecule inhibitor, trifluoperazine (TFP), induces changes in atomic arrangement and hydration that leads to yet another unique chemical entity (Fig. 2D) (67). These graphical representations, generated from X-ray structure found in the Protein Data Bank [Research Collaboratory for Structural Bioinformatics (RCSB)/Protein Data Bank (PDB)], for bound and unbound species pictorially illustrate that the potential magnitude of free-solution signal can be large (approximately >10 −4 RIU) under the proper conditions.…”

Section: Conformation and Hydration Changes Are The Origin Of Free-somentioning

confidence: 99%

Origin and prediction of free-solution interaction studies performed label-free

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...

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“…However, the data available thus far are still insufficient to provide a complete picture of the functions of CaM during pollen tube development. TFP is a potent antipsychotic phenothiazine and has been widely studied in relation to its mode of binding and specific inactivation of CaM (Picton and Steer, 1985;Vandonselaar et al, 1994;Krinke et al, 2007). Therefore, we set up several pharmacological experiments including large-scale proteomic analysis using safe concentrations of TFP in order to extend our knowledge of the involvement of CaM in pollen tube growth.…”

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confidence: 99%

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

Chen

et al. 2008

Plant Physiology

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Ca 2+ -calmodulin (Ca 2+ -CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca 2+ -CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca 2+ -CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca 2+ -CaM signaling rapidly induced an increase in extracellular Ca 2+ influx, resulting in dramatically increased cytosolic Ca 2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca 2+ -CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca 2+ -CaM in the regulation of tip growth machinery.

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Trifluoperazine-induced conformational change in Ca2+-calmodulin

Cited by 213 publications

References 37 publications

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Origin and prediction of free-solution interaction studies performed label-free

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

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