Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Abstract

Background Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. Results We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. Conclusion Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Graphical abstract Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Identification and validation of autophagy-related genes in primary open-angle glaucoma

“…The results proved that the expression pro les of drug disturbances such as Calmidazolium, Cephaeline, Emetine and Vinorelbine were most signi cantly negatively correlated with the expression pro les of disease disturbances. Calmidazolium is a calmodulin inhibitor [79]. Both Cephaeline and Emetine are pleiotropic alkaloids isolated from ipecacuanha trees, and the difference is that Emetine contains a methoxy group while Cephaeline contains a hydroxyl group.…”

Phytochemical Interactions with Calmodulin and Critical Calmodulin Binding Proteins Involved in Amyloidogenesis in Alzheimer’s Disease

“…Two pairs of calcium-binding EF-hands, the N-terminal and C-terminal lobes are connected via a flexible linker ( Figure 2 ). It is this binding domain flexibility that endows CaM with its ability to bind to and regulate such a diversity of proteins, many of which are involved in the onset and progression of AD [ 6 , 23 ]. In the presence of calcium ions, a conformational change exposes methionine-rich hydrophobic pockets that permit binding to target proteins via hydrophobic amino acid-rich CaM-binding domains (CaMBDs).…”

“…On the other hand, apo-CaM binds to a smaller population of calcium-independent CaMBPs via IQ [FILV]Qxxx[RK]Gxxx[RK]xx[FILVWY]; IQ-like [FILV]Qxxx[RK]Gxxxxxxxx); IQ-2A [IVL]QxxxRxxxx[VL][KR]xW; IQ-2B [IL]QxxCxxxxKxRxW and IQ variant [IVL]QxxxRxxxx[RK]xx[FILVWY] domains [ 20 ]. The chemical structure of CaM antagonists varies but routinely they are typically hydrophobic molecules with a net positive charge, as exemplified by calmidazolium binding, allowing them to bind to the hydrophobic patches and pockets of Ca 2+ /CaM thereby interfering with the binding and activation of target CaMBPs [ 23 ].…”

New Insights into the Regulation of mTOR Signaling via Ca2+-Binding Proteins

“…An identical CaM protein is encoded by three genes ( CALM1 , CALM2 , and CALM3 ) in mammals. The binding of Ca 2+ to CaM induces a significant conformational change from a closed to an open state, exposing a hydrophobic surface that facilitates the binding with its target protein [ 58 , 59 ]. CaM plays vital roles in a wide range of biological processes, including cell growth [ 60 ], cell cycle progression, proliferation [ 61 , 62 ], and trafficking [ 63 ] by binding to hundreds of target proteins in a Ca 2+ -dependent manner.…”