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

¹ Departamento de Química Inorgánica, Analítica y Química-Física e Instituto de Química Física de los Materiales, Medio Ambiente y Energia, Consejo de Investigaciones Científicas y Técnicas (INQUIMAE-CONICET), Facultad de Ciencias Exactas y Naturales and ² Laboratorio de Neuroquímica Retiniana y Oftalmología Experimental, Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
³ Molecular Structure Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom
⁴ Consiglio Nazionale delle Ricerche, Istituto di Biofisica (Palermo) (CNR-IBF), I-90147, Palermo, Italy

(RECEIVED January 5, 2004; FINAL REVISION March 22, 2004; ACCEPTED May 8, 2004)

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca²⁺ 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 ¹⁵N and ¹H 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 Ca²⁺ by paramagnetic Tb³⁺ 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.

Keywords: melatonin; calmodulin; molecular dynamics; NMR; fluorescence; weak interactions

Abbreviations: CaM, calmodulin • C-CaM, C-domain of calmodulin • NMR, nuclear magnetic resonance • HSQC, heteronuclear single quantum coherence • NOESY, nuclear Overhauser effect spectroscopy • TOCSY, total correlation spectroscopy • TFP, trifluoperazine • J-8, N-(8-aminooctyl)-5-iodonaphthalene-1-sulfonamide • W-7, N-(6-aminhexyl)-5-chloro-1-naphthalenesulfonamide • AAA, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine • Mel, melatonin • MD, molecular dynamics • FEP, free energy perturbation • SM0, MD run of isolated Mel • SM1, MD run of Mel in solution • SC1, MD run of C-CaM in solution • SMC1, MD run of the C-CaM-Mel complex • SMC2, FEP run of the C-CaM-Mel complex

Reprint requests to: Vincenzo Martorana, CNR-IBF, Istituto di Biofisica (Palermo), via U. La Malfa 153, I-90147, Palermo, Italy; e-mail: vincenzo.martorana{at}pa.ibf.cnr.it; fax: +390916809349.

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