NMR and molecular dynamics studies of the interaction of melatonin with calmodulin

Adrián G Turjanski,Vincenzo Martorana,Rodolfo R Biekofsky,Ruth E Rosenstein,Annalisa Pastore,Darío A Estrin,John E Mccormick,Stephen R Martin

doi:10.1110/ps.04611404

Abstract

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca(2+) concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring (15)N and (1)H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca(2+) by paramagnetic Tb(3+) allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.

Highlights

It has been suggested that rat hypothalamic and striatal nitric oxide synthase (NOS) inhibition is regulated by the interaction of melatonin and some melatonin-related kynurenines with CaM (Pozo et al 1997; León et al 2000), in the hamster retina NOS inhibition seems to be independent of CaM (Saenz et al 2002)
Fluorescence intensity measurements were used to study the interaction of CaM with melatonin following the approach used previously by other researchers (Ouyang and Vogel 1998)
In our opinion, the need for new, more reliable methods to estimate the absolute free energy of binding (Luo et al 2002). This is especially true in weakly interacting complexes in which there are no specific interactions, such as the case for melatonin–calmodulin. Both experimental and molecular dynamics (MD) results show that the calciumdependent interaction of melatonin with CaM is weak

Summary

Introduction

Hydrophobic molecules bind to CaM and modify its function by inhibiting the interaction with other proteins (Prozialeck and Weiss 1982; Weiss et al 1985; Cook et al 1994; Vandonselaar et al 1994; Craven et al 1996; Osawa et al 1998; Harmat et al 2000) Some of these compounds used as pharmacological agents to block CaM-mediated enzyme activation may act as antipsychotics, muscle relaxants, antidepressants, minor tranquilizers, and local anesthetics (Weiss et al 1982; Zhang et al 1990).

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Conclusion