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

Menyhárd, Dóra K.; Keserű, György M; Náray‐Szabó, Gábor

doi:10.2174/157340909789577874

Cited by 14 publications

(11 citation statements)

References 108 publications

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“…There are two main classes of calmodulin residues that are consistently described as participating in binding events with target peptides. These are either hydrophobic residues such as Phe, Leu, or Met in so-called FLMM cavities in both the N- and C-terminal calmodulin lobes (where the letters stand for the comprised amino acids) or charged Glu residues surrounding the FLMM cavities [ 34 ]. The FLMM residues anchor the ligand through conserved hydrophobic interactions and the glutamic acid sidechains stabilize the interaction through electrostatic contacts [ 35 , 36 ].…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

et al. 2015

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Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.

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Section: Resultsmentioning

confidence: 99%

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

et al. 2015

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“…Table S2 shows the interface residues involved in each complex formation, where most of the residues are hydrophobic character. In the case of CaM, this is not new because the X-ray structure of Ca 2+ -CaM bound to different targets has revealed CaM hydrophobic patches (Yang et al, 2004;Menyhard et al, 2009).…”

Section: Protein-protein Dockings and Determination Of Interfaces In mentioning

confidence: 99%

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

González-Andrade

Mata

Madariaga-Mazón

et al. 2013

J of Molecular Recognition

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Protein-protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein-protein-ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII-spectrin peptide (αII-spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C-mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM-protein complex under analysis. For the Ca(2+) -CaM, Ca(2+) -CaM-PDE1A, and Ca(2+) -CaM-MLCK complexes, CPZ apparent dissociation constants (Kds ) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII-spec) to Ca(2+) -hCaM M124C-mBBr quenched the fluorescence (Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII-spec to a preformed Ca(2+) -hCaM M124C-mBBr-MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca(2+) -CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca(2+) -CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands.

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“…During the calcium-binding process, it changes from a closed structure to an open structure, exposes its hydrophobic cleft and is then ready to dock to and modulate other important proteins. This ion-induced dynamic behavior has been studied deeply for a long time [18], from ion-binding affinity [19][20][21], domain movement [22][23][24][25] to calcium-protein docking [26][27][28][29][30][31]. In this paper, we just pay attention to the ion-binding affinity of its four EF-hand loops.…”

Section: Introductionmentioning

confidence: 99%

Binding free-energy calculation of an ion-peptide complex by constrained dynamics

et al. 2013

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Binding free energy is the most important physical parameter that describes the binding affinity of a receptor-ligand complex. Conventionally, it was obtained based on the thermodynamic cycle or alchemical reaction. These strategies have been widely used, but they would be problematic if the receptors and/or ligands have large conformational changes during the binding processes. In this paper, we present a way to calculate the binding free energy: constrained dynamics along a fragmental and high-dimensional transition path. This method directly considers unbound states in the simulation. The application to the calmodulin loop-calcium complexes shows that it is practical and the calculated relative binding affinities are in good agreement with experimental results.

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

Cited by 14 publications

References 108 publications

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

Identification and Characterization of the Interaction Site between cFLIPL and Calmodulin

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Binding free-energy calculation of an ion-peptide complex by constrained dynamics

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