Markov state modelling reveals heterogeneous drug-inhibition mechanism of Calmodulin

Calmodulin (CaM) is a calcium sensor which binds and regulates a wide range of target-proteins. This implicitly enables the concentration of calcium to influence many downstream physiological responses, including muscle contraction, learning and depression. The antipsychotic drug trifluoperazine (TFP) is a known CaM inhibitor. By binding to various sites, TFP prevents CaM from associating to target-proteins. However, the molecular and state-dependent mechanisms behind CaM inhibition by drugs such as TFP are largely unknown. Here, we build a Markov state model (MSM) from adaptively sampled molecular dynamics simulations and reveal the structural and dynamical features behind the inhibitory mechanism of TFP-binding to the C-terminal domain of CaM. We specifically identify three major TFP binding-modes from the MSM macrostates, and distinguish their effect on CaM conformation by using a systematic analysis protocol based on biophysical descriptors and tools from machine learning. The results show that depending on the binding orientation, TFP effectively stabilizes features of the calcium-unbound CaM, either affecting the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the bound domain. The conclusions drawn from this work may in the future serve to formulate a complete model of pharmacological modulation of CaM, which furthers our understanding of how these drugs affect signaling pathways as well as associated diseases. Author summary Calmodulin (CaM) is a calcium-sensing protein which makes other proteins dependent on the surrounding calcium concentration by binding to these proteins. Such protein-protein interactions with CaM are vital for calcium to control many physiological pathways within the cell. The antipsychotic drug trifluoperazine (TFP) inhibits CaM's ability to bind and regulate other proteins. Here, we use molecular dynamics simulations together with Markov state modeling and machine learning to understand the structural and dynamical features by which TFP bound to the one domain of CaM prevents association to other proteins. We find that TFP encourages CaM to adopt a conformation that is like the one stabilized in absence of calcium: depending on the binding orientation of TFP, the drug indeed either affects the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the domain. Understanding TFP binding is a first step towards designing better drugs targeting CaM.

Automated summary

Calmodulin (CaM) is a calcium-sensing protein that regulates other proteins by binding them in a calcium-dependent manner. Trifluoperazine (TFP), an antipsychotic drug, inhibits the ability of CaM to bind and regulate other proteins. In this study, we used molecular dynamics simulations, Markov state modeling, and machine learning to understand how TFP binding to one domain of CaM prevents its association with other proteins. We found that TFP promotes the adoption of a conformation similar to the calcium-unbound form of CaM, affecting different structural and dynamic features depending on its binding orientation. Understanding TFP binding is a crucial step towards developing improved drugs that target CaM.