| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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
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.
(RECEIVED January 5, 2004; FINAL REVISION March 22, 2004; ACCEPTED May 8, 2004)
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
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
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Small, 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). CaM antagonists (such as trifluoperazine [TFP] and many others) are also being evaluated for the treatment of cancers that are both sensitive and resistant to conventional anticancer drugs (Ford and Hait 1990; Liu et al. 2002), because diseases characterized by unregulated cell growth have been shown to present elevated levels of Ca2+-CaM (Hickie et al. 1983; Dai et al. 2002).
One of the endogenous CaM antagonists is thought to be the pineal hormone melatonin (N-acetyl-5-methoxytryptamine) (Fig. 1
; Benitez-King et al. 1993 , 1996; Cardinali et al. 1997). Melatonin is involved in several physiological functions, including the entrainment of both seasonal and circadian rhythms. It has been shown that melatonin prevents the Ca2+/CaM inhibition of microtubule polymerization both in situ and in vitro assays (Huerto-Delgadillo et al. 1994). More recently, it has been suggested that rat hypo-thalamic 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), although in the hamster retina NOS inhibition seems to be independent of CaM (Saenz et al. 2002).
|
In the present paper, conclusive atomistic evidence of the CaM/melatonin interaction is provided by fluorescence and nuclear magnetic resonance (NMR) studies, and by molecular dynamics (MD) simulations. NMR chemical shift mapping shows a significant and reversible binding of melatonin to both domains of CaM. Molecular docking and MD simulations provide a further atomistic characterization of the CaM–melatonin complex.
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
). Because the interaction is weak (Kd > 2 mM) it was not possible to determine a precise value of the dissociation constant or to establish the stoichiometry of the interaction. A control titration with Ca2+-free CaM (apo-CaM) showed no change in fluorescence intensity, showing that the interaction is calcium-dependent.
|
shows the superposition of 1H-15N HSQC spectra taken at different [melatonin]/[CaM] molar ratios, showing the progressive shift of peaks corresponding to selected HN signals during the titration. Figure 3B shows changes in 1H and 15N chemical shifts observed upon complexation of CaM with melatonin for some representative residues.
|
of 5 mM vs. 
of 7 mM, Table 1). As expected for such weak binding contacts, no plateau could be reached in the titration even at high CaM–melatonin ratios, and no intermolecular NOE effects could be observed.
|
, A and B, show the 1H and 15N chemical shift differences between [melatonin]/[CaM] molar ratios of 0 and 9 for all residues in CaM. Figure 4C presents a weighted average of the chemical shift changes observed for each residue, and gives a rough indication of the extent to which a residue is affected by complexation with melatonin. Complex formation significantly affects ~22% of the CaM resonances. The most significant changes are localized mainly in the hydrophobic pockets of either domain (Fig. 4D).
|
As shown in previous work, the Tb3+ added binds preferentially to the N-domain of CaM (Biekofsky et al. 1999). The binding of Tb3+ ions to the N-domain gives rise to three paramagnetic species, according to whether Tb3+ binds separately to sites I or II, or simultaneously to both these sites. For each amide NH group there will be up to four signals in an 1H-15N HSQC spectrum: the signal corresponding to the (Ca2+)4 species, and three signals corresponding to Tb3+(I)–(Ca2+)3, Tb3+(II)–(Ca2+)3, and Tb3+(I)–Tb3+(II)–(Ca2+)2–calmodulin (where I and II indicate the binding sites of the N-domain, III and IV those of the C-domain). Figure 5 shows the four peaks corresponding to C-domain residues Ile 100 and Val 136. Such patterns are seen throughout the spectrum for many residues. However, in crowded areas of the HSQC spectrum this leads to superposition of peaks, making the RDC and PCS measurement difficult. For residues very near to the Tb3+ ions, the NMR signals of Tb3+ species become broad and cannot be observed (mostly those in the N-domain). Such is the case, for example, of the N-domain residue Ile 27 shown in Figure 5.
|
presents data measured for selected residues in these three systems to provide examples of the range of values obtained. The residues included in Table 2 were chosen from both domains, and show examples of both positive and negative pseudocontact shifts, and positive and negative dipolar coupling contributions to the NH splittings. For each residue shown in Table 2, the data measured for the four species in solution are given, except for the N-domain residues such as Gly 33 or Ala 57, for which the signals corresponding to some species could not be found.
|
There is evidence that other small molecules binding to Ca2+/CaM induce interdomain compaction. For instance, the binding of two W-7 molecules to Ca2+/CaM in solution induces a globular shape for Ca2+/CaM, probably caused by the collapse of both domains (Osawa et al. 1999). As well, the crystal structures of Ca2+/CaM-(TFP)1 (Cook et al. 1994), Ca2+/CaM-(TFP)2 (Vertessy et al. 1988), and Ca2+/CaM-(TFP)4 (Vandonselaar et al. 1994) reveal juxtaposition of both CaM domains compared with the dumbbell structure of Ca2+/CaM (Babu et al. 1988). In these structures, a hydrophobic tunnel resulting from the juxtaposition of the hydrophobic patches of the N- and C-terminal domains of CaM is very similar to that seen in the peptide complexes. In the present work, it is important to make the case of autonomy of the domains in the CaM–melatonin complex, which allows us to use the strategy of characterizing computationally the binding of melatonin to individual domains.
MD of uncomplexed CaM
Based on the independent behavior of the two homologous domains in the CaM:melatonin complex, and to limit the computational load, we decided to restrict the MD simulations to the C-terminal domain. A MD trajectory (SC1 trajectory) was calculated for C-CaM. The root mean square displacement (RMSD) of the backbone 
-carbon atoms of C-CaM and C-CaM:melatonin with respect to the initial structure is shown in Figure 6. Global translational and rotational motions of the protein were removed by least square superposition of the -carbon atoms relative to the initial structure. The overall protein structure was found to be stable, because the RMSD values are of the order of 1 Å for C-CaM. Our results are consistent with simulations of full CaM solvated by a water cluster (Wriggers et al. 1998). In the C-CaM:melatonin complex the RMSD values are bigger (see below).
|
MD simulations of the complex melatonin–CaM
In the course of a 2.8-nsec MD simulation (SMC1 trajectory) of the melatonin–C-CaM complex, the melatonin position with respect to C-CaM exhibited large fluctuations. We have selected four geometrical parameters to structurally characterize the interaction. The first two parameters are meant to represent the rotation of the melatonin heterocycle with respect to the initial orientation. We have defined two quasi-orthogonal axes: One is defined by the two centers of mass of each ring of the indole moiety, and the other one is the axis that goes along the bond that connects the two carbon atoms (atoms 10 and 12 of Fig. 1
) linking the two rings. The angle of these axes with respect to their orientation in the first snapshot of the simulation is measured along the trajectory (Fig. 7B). These angles give information about the rotation of the melatonin heterocycle. We also measured the distance of the indolic nitrogen, NA, and the methoxy oxygen, OE (atoms 9 and 16, respectively, in Fig 1), to the geometrical center determined by the -carbon atoms of Ile 100 and Val 136 (Fig. 7A). These residues were chosen because they are located at the bottom of the hydrophobic pocket and their positions fluctuate very little during the simulation. Global translational and rotational motions of the protein were removed by least-square superposition of the -carbon atoms relative to the initial structure. The time evolution of these parameters is presented in Figure 7B.
|
, A–C. Due to the limited time of our simulation, it may be possible that we have not sampled other relevant configurations.
|
). This configuration is very similar to that exhibited by the MLCK complex (Meador et al. 1992; see below). The amino acids whose average distance from the ligand, during the initial 0.2 nsec of simulation SMC1 is less than 6 Å, are shown in Table 3. It can be seen that they all belong to the hydrophobic pocket. To compare with other complexes of calmodulin the distances of the calmodulin amino acids to TRP(MLCK), TFP and W7 in their X-ray structures are also shown in Table 3. Interestingly, similar amino acids are found in the different complexes, indicating that the interaction with the C-terminal hydrophobic pocket seems to be an invariant of the CaM recognition: the two residue 
-sheet side chains (Ile 100 and Val 136), the aromatic cluster (Phe 92, Phe 141), the aliphatic cluster (Ile 105, Ile 125, and Ala128), and the four methionine residues (Met 109, Met 124, Met 144, and Met 145). However, there is a difference in the W7 complex with respect to the other complexes: Amino acids 82 to 87 are found near the W7 ligand, at odds with other cases. Probably, this is due to the fact that W7 has a long positive chain interacting with residues Glu 82, Glu 83, Glu 84, and Glu 87. This tail is lacking in melatonin, while in the TRP(MLCK) case there are other amino acids with positive groups interacting with the negative glutamates, and there are other TFP molecules interacting with the negative groups of calmodulin in the 1:4 complex with TFP. This lack of a positive coil could be the reason for the weaker interaction of melatonin.
|
), that get as large as 12 Å and 15 Å, respectively. The indolic ring gets also parallel to helices 1 and 3, rotating both axes about 150° with respect to the initial structure. At about 0.6 nsec melatonin gets inside the hydrophobic pocket, but with a different orientation corresponding to a rotation of 100° of both axes, compared to the initial docking structure. The NA and OE distances are similar to those found in the first structure. This configuration, depicted in Figure 8C, is found with little variations, until the end of the simulation. Analysis of the changes in energy contributions (internal, electrostatic, van der Waals) during the trajectory (not shown) suggests that a major role in determining melatonin position and conformation is played by electrostatic interaction with protein and with solvent.
Flexibility of C-CaM along the simulation
C-CaM complexed with melatonin exhibits large structural fluctuations along the simulation, as shown in Figure 6. To compare with the SC1 simulation we have again calculated the RMSD of the -carbon backbone atoms with respect to the initial structure. Global translational and rotational motions of the protein were removed by least-square superposition of the -carbon atoms relative to the initial structure. The average RMSD starts to oscillate around a value of about 2 Å and then increases to values around 4 Å. A more detailed analysis shows that large displacements are limited, apart from the terminations, to the coil that links helix 2 to helix 3, to helix 3 itself, and to the second loop (numbering of helices is made considering the C domain of CaM only). This is probably due to the fact that during the simulation melatonin changes its position with respect to calmodulin significantly, even leaving the hydrophobic pocket.
Notwithstanding the large RMSD, the secondary structure of C-CaM is well conserved along the Molecular Dynamics trajectory, preserving a well-defined hydrophobic pocket. Indication of major conformational change of CaM has been found in the case of binding to trifluoroperazine (Vandonselaar et al. 1994). In this case, the movement involves the bending of the central helix that links the two domains, but small changes have been observed in the C-and N-terminal domains in the molecular dynamics simulations of the 4TFP:CaM (Yamaotsu et al. 2001). This is in agreement with a stronger interaction between the drug and the protein resulting in small fluctuations of the TFP position. The occurrence of large concerted motions of C-CaM in the presence of melatonin may raise some doubts about the validity of the docking procedure, where a single protein configuration was considered. Actually, the importance of the hydrophobic pocket is confirmed both by direct experimental evidence (this work) and crystal structures of similar complexes (see Discussion).
Free energy of binding
In addition to the above MD simulations, a theoretical estimation of 
G° associated to the binding process was also performed, following the double annihilation method (Jorgensen et al. 1988). The result G° = –9 ± 0.4 kcal/mole, corresponding to Kd less than micromolar, is far from the experimental one (–3.2 ± 0.2 kcal/mole) and at odds with Molecular Dynamics results, where melatonin is seen to exit easily from the pocket. The reasons for such disagreement are (1) the poor sampling of relevant configurations of the complex, notwithstanding the considerable length of simulation SMC2 (15 nsec); (2) the force-field inaccuracies; and (3) the flaw intrinsic to the method (Gilson et al. 1997). Our computation confirms, 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.
|
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Chemical shift perturbation allowed us to map the interaction in the hydrophobic cavities of each of the CaM globular domains. Based both on this evidence and on the experimental information that the two globular domains retain their orientational freedom in the complex, we chose to model the CaM–melatonin complex by following the dynamical behavior of one melatonin molecule in the pocket of the C-terminal domain. The most stable complex found by molecular docking, shows melatonin inside the hydrophobic pocket, with the O-methyl group pointing to the bottom of the pocket and the ethylamide side chain located in the entrance. The aromatic ring of melatonin is relatively immobilized, with the aliphatic tail extended into the solvent. Two main orientations were found for this arrangement, and within each orientation the ring retains some degree of translational freedom.
In the course of our MD simulations, melatonin explores different conformations, in some cases, even leaving the hydrophobic pocket. Melatonin binds to C-CaM mainly by hydrophobic interactions of the indolic moiety with the aliphatic and aromatic amino acids of the C-CaM hydrophobic pocket. However, from about 0.6 nsec to 2 nsec the indolic nitrogen also forms a hydrogen bond with the carbonyl of Ala 128. This is consistent with previous results which suggested an important role for Ala 128 in ligand binding to CaM (Osawa et al. 1998).
The main average structural features obtained for the melatonin–CaM complex are in excellent agreement with those of other CaM complexes formed both with peptides and with small molecules (Fig. 9). The interaction with the C-terminal pocket seems to be an invariant of the CaM recognition: In all available structures, a bulky hydrophobic residue binds inside the CaM pocket of the C-domain. In CaM–peptide complexes, this site is often occupied by a tryptophan. A second hydrophobic site anchors the molecule to the N-terminal domain, helped by the additional support of a basic group, which interacts with residues outside the pocket (Prozialeck and Weiss 1982; Weiss et al. 1985), thus resulting in a strong binding constant. Comparison of the CaM complexes with small molecules both obtained by crystallographic studies, such as those of TFP (PDB: 1CTR
[PDB]
, 1A29
[PDB]
, 1LIN
[PDB]
), or DPD (PDB: 1QIV
[PDB]
), or by NMR, such as those of W7 (PDB: 1MUX
[PDB]
), also shows that the C-terminal hydrophobic pocket is always occupied. Additional binding sites may be present outside the pocket, but mostly sandwiched in between the two domains (as is the case of 1LIN
[PDB]
).
|
Biological implications
Both melatonin and CaM are ubiquitously found and their structures are phylogenetically well preserved. The fact that melatonin interacts weakly with CaM, does not detract the possibility that this interaction may occur in biological media. The weak melatonin–CaM interaction may allow the hormone to gently modulate many cellular functions in a rhythmical fashion. The cyclic rise in melatonin bloodstream levels stimulated by darkness would have the effect of producing a subtle inhibition of CaM’s interaction with other proteins. This inhibition has been shown in vitro for certain systems (Benitez-King et al. 1993; Cardinali et al. 1997; León et al. 2000), and remains, however, difficult to assess in vivo. A higher affinity for the CaM–melatonin interaction would probably render this mechanism unsuitable.
On the other hand, the formation of the complex in biological media will depend on the concentrations of both species in different physiological microenvironments within the different cells. In fact, while this interaction was observed in some tissues (Benitez-King et al. 1996; Pozo et al. 1997; León et al. 2000), it was not confirmed in other systems (Wolfler et al. 1998; Sáenz et al. 2002). Melatonin may accumulate in some tissues and cellular structures, such as cell membrane, cytosol, and nuclei, to about 103 times higher than circulating levels (Menendez-Pelaez et al. 1993). The affinity measured for this interaction, although low, is able to explain in vitro effects that have been observed in previous papers (Pozo et al. 1997; León et al. 2000).
Moreover, from the pharmacological point of view, melatonin has been found to be an important anticancer agent. The proposed mechanism of action of this drug is via its inhibition of Ca2+/calmodulin, and because pharmaceutical dosage of melatonin is high (but not toxic) the low Kd of the interaction is not a problem (MacNeil et al. 1988). The therapeutic use of melatonin as well as its safety, dosage, side effects, and contraindications have been extensively investigated (e.g., see Karasek et al. 2002). The drug has been shown to present a very low toxicity (Karasek et al. 2002), and therefore, it can be considered as a viable pharmacological alternative to other more toxic antagonists for inhibiting CaM activity.
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Fluorescence spectroscopy
Uncorrected fluorescence emission spectra were recorded using a SPEX Fluoromax fluorimeter with 
ex = 295 nm (bandwidth 0–85 nm) and emission from 310 to 450 nm (bandwidth 5 nm). Spectra were recorded at 20°C in 25 mM Tris (pH 8), 100 mM KCl with 1 mM CaCl2 and 1 mM EGTA, as appropriate.
NMR spectroscopy
All NMR spectra were recorded at 500 and 600 MHz (1H frequency) and at 35°C on Varian Unity Plus 500 and 600 spectrometers. Two-dimensional 15N-1H HSQC (Bodenhausen and Ruben 1980; Mori et al. 1995) spectra, with WATERGATE sequence (Piotto et al. 1992; Sklenár et al. 1993), were acquired at 500 MHz with spectral widths of 7500 Hz (1H) and 2027 Hz (15N), respectively. A total of 110 t1 increments were acquired with an acquisition time of 0.15 sec, during which the 15N nuclei were decoupled using a GARP sequence (Shaka et al. 1985).
Assignment of 1H and 15N resonances for Ca2+-CaM was taken from Ikura and coworkers (Ikura et al. 1990), and confirmed by three-dimensional 15N-1H NOESY-HSQC and TOCSY-HSQC experiments (Marion et al. 1989; Zuiderweg and Fesik 1989). A 15N-1H NOESY-HSQC was acquired at 500 MHz with spectral widths as above, a mixing time of 100 msec, 125 increments in the indirect 1H dimension and 48 increments for the 15N dimension. A 15N-1H TOCSY-HSQC (Marion et al. 1989) was acquired at 600 MHz with spectral widths of 7996 Hz (1H) and 2431 Hz (15N), a mixing time of 60 msec, 130 increments in the indirect 1H dimension and 40 increments for the 15N dimension. Acquisition times of 0.15 sec were used throughout, again with decoupling of the 15N nuclei using a GARP sequence.
Residual dipolar couplings were extracted from 2D IPAP 1H-15N HSQC spectra (Ottiger et al. 1998). Spectra were acquired with 1184 complex points in t2 (148 msec acquisition time) with a spectral width of 8000 Hz, and 270 complex points were collected in t1 (108 msec acquisition time) with a spectral width of 2500 Hz.
All spectra were processed using nmrPipe (Delaglio et al. 1995) and analyzed using XEASY (Bartels et al. 1995). Typically, the acquisition dimension was multiplied by a Gaussian function, and other dimensions with a 90°-shifted sine-bell function. All dimensions were zero-filled at least to the next power of 2. Chemical shifts of 1H resonances were measured from an internal standard dioxane and referenced to 5,5-dimethyl-5-silapentane-2-sulphonate (taken as 3.750 ppm from dioxane) (Wishart et al. 1995), 15N chemical shifts are referenced to liquid NH3 using the frequency ratio method (15N/1H = 0.101329118) (Wishart et al. 1995).
Melatonin titration
Melatonin was purchased from Aldrich Chemical Co. Ltd. A stock solution of 13 mM melatonin in 90% H2O/10% D2O was prepared. For each titration point an aliquot of 50 µL of this stock melatonin solution was added to the NMR tube containing the CaM solution and mixed thoroughly. The changes in pH caused by melatonin additions were negligible.
2D 1H-15N HSQC NMR spectra were recorded for 15N-labeled (Ca2+)4-CaM at various melatonin concentrations from [melatonin]/[CaM] molar ratio of 0 up to 9. The signals in the spectra are well-resolved and individual amide NH 1H/15N signals can be followed throughout the titration. The previously assigned 1H and 15N signals for Ca2+-saturated CaM (Ikura et al. 1990) were used to identify signals in the 2D 1H-15N HSQC spectrum for the [melatonin]/[CaM] molar ratio of 0. Any ambiguities in the assignment of amide NH 1H/15N resonances during the melatonin titration were resolved by using three-dimensional TOCSY-HSQC and NOESY-HSQC experiments.
Partial replacement of Ca2+ by Tb3+
A stock solution of 50 mM TbCl3 in 90% H2O/10% D2O was prepared from a standard 1 M TbCl3 solution; 8.64 µL of this stock solution was added to the solution of the CaM:melatonin complex and mixed thoroughly giving a [Tb3+]/[CaM] molar ratio of 0.66 eq of the CaM concentration. The changes in pH caused by TbCl3 additions were negligible.
Analysis of binding curves
The chemical shift variation of individual resonances were fitted using the following equation: 
obs = i + [f – i] • [bound melatonin]/[total CaM], where i and f are the initial and final CaM chemical shifts, [bound melatonin] is the concentration of bound melatonin, and [total CaM] is the total concentration of CaM. Fits were performed using standard nonlinear least-squares methods. To determine Kd values from the NMR experimental data, we are assuming a linear relation between complexation and chemical shift changes.
Computational tools
Melatonin force field parametrization
Partial charges for melatonin were obtained using the restrained electrostatic fit (RESP) scheme (Breneman and Wiberg 1990). Electrostatic potentials were computed using ab initio HF/6–31G** calculations using the Gaussian 98 package (Frisch et al. 1998). Most of the parameters for melatonin were taken from the AMBER force field (Cornell et al. 1995). The parameters associated with the torsional degrees of freedom of the ethyl amide chain were not available and were obtained by fitting them iteratively to reproduce the energy profiles obtained from HF/6–31G** calculations (Cornell et al. 1995). The atom types and force field parameters for melatonin are given as supplementary material.
MD simulations
MD simulations were performed using the Amber force field for CaM (Cornell et al. 1995), and the TIP3P model for water (Jorgensen et al. 1983). Calcium ions were represented using the parameters derived by Hori and collaborators (Hori et al. 1988), while parameters for melatonin are described above. Apart from the preparatory work, for which we used AMBER6 software package (Case et al. 1999), all simulations were performed using the package NAMD (Kale et al. 1999).
In all cases, the time step was 2 fsec, the van der Waals interactions were smoothly cut off at 9 Å, while electrostatic interactions were accounted for by using the particle mesh Ewald method (Essman et al. 1995). All bonds were kept rigid (Ryckaert et al. 1977). The Berendsen thermostat (Berendsen et al. 1984) was used to maintain the desired temperature.
Simulations of melatonin
The initial coordinates of melatonin were taken from the HF/6–31G** calculations. The temperature was set to 310 K. Aqueous simulations were performed by placing melatonin into a periodic box consisting of 803 TIP3P water molecules. The volume of the box was adjusted to obtain a pressure of 1 atm using the Berendsen method with a relaxation time of 1 psec (Berendsen et al. 1984). Two trajectories, of 1 and 4 nsec each, were respectively obtained for the isolated (SM0) and solvated melatonin (SM1 trajectory).
Simulations of C-CaM
The crystal structure from the Brookhaven Protein Data Bank (PDB) 4CLN
[PDB]
(Taylor et al. 1991) was taken as the initial structure of CaM. The initial positions of the hydrogen atoms were generated by the LEAP module of the AMBER package (Case et al. 1999). The C-terminal domain of CaM (residues 81–148) was placed into a periodic box consisting of 3525 TIP3P water molecules. To neutralize the system, eight potassium ions were also included in the simulation. After an equilibration phase we obtained 1-nsec MD trajectory (SC1 trajectory) at 310 K and 1 atm.
Molecular docking of melatonin:C-CaM complex
Docking simulations were performed with the AUTODOCK 3.0 program (Goodsell and Olson 1990; Morris et al. 1998). The CaM and melatonin initial structure were taken from the SC1 and SM1 MD trajectories, respectively. Lennard-Jones parameters and partial charges were assigned according to the atom types, consistently with the AMBER force field (Cornell et al. 1995). A mesh with a spacing of 0.55 Å was defined in a box measuring 50 x 50 x 50 Å3 around C-CaM. The energy of interaction between C-CaM and melatonin were computed in each point of the grid. The torsional modes associated with ethyl amide and the methoxyl moieties of melatonin were left free while the rest of the molecule was considered rigid.
A Monte Carlo simulated annealing scheme was employed to locate relevant minima. The initial temperature was set to 503 K. We performed 50 Monte Carlo runs, each comprising 50 cooling cycles with a scaling factor of 0.9. The final temperature was 2.6 K.
MD simulations of melatonin:C-CaM complex
MD complex simulations were performed, starting with the most stable structure found with the docking protocol described above. The complex was neutralized with eight potassium ions, and was placed in a periodic box consisting of 3128 water molecules at 300 K and 1 atm. The simulation consisted of a 2.8-nsec trajectory (SMC1).
The theoretical melatonin:C-CaM binding constant was estimated using the free energy perturbation method (Kollman et al. 1993) and the so-called double annihilation process (Jorgensen et al. 1988). The free energy change associated with the binding process was computed by simulating the transfer of melatonin from the complex and from solution to the gas phase (simulations SMC2 and SM2, respectively).
|
Footnotes |
|---|
Supplemental material: see www.proteinscience.org
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W., and Bax, A. 1992. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: The central helix is flexible. Biochemistry 31: 5269–5278.[CrossRef][Medline]
Bartels, C., Xia, T., Billeter, M., Güntert, P., and Wüthrich, K. 1995. The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 5: 1–10.[CrossRef][Medline]
Benitez-King, G., Huerto-Delgadillo, L., and Anton-Tay, F. 1993. Binding of 3H-melatonin to calmodulin. Life Sci. 53: 201–207.[CrossRef][Medline]
Benitez-King, G., Ríos, A., Martine, S., and Anton-Tay, F. 1996. In vitro inhibition of Ca2+/calmodulin-dependent kinase II activity by melatonin. Biochim. Biophys. Acta 1290: 191–196.[Medline]
Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., Di Nola, A., and Haak, J.R. 1984. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81: 3684–3690.[CrossRef]
Berridge, M.J., Bootman, M.D., and Lipp, P. 1998. Calcium—A life and death signal. Nature 395: 645–648.[CrossRef][Medline]
Biekofsky, R.R., Martin, S.R., Browne, J.P., Bayley, P.M., and Feeney, J. 1998. Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual site Ca2+ coordination. Biochemistry 37: 7617–7629.[CrossRef][Medline]
Biekofsky, R.R., Muskett, F.W., Schmidt, J.M., Martin, S.R., Browne, J.P., Bayley, P.M., and Feeney, J. 1999. NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+. FEBS Lett. 460: 519–526.[CrossRef][Medline]
Blask, D.E., Sauer, L.A., and Dauchy, R.T. 2002. Melatonin as a chronobiotic/anticancer agent: Cellular, biochemical, and molecular mechanisms of action and their implications for circadian-based cancer therapy. Curr. Top. Med. Chem. 2: 113–132.[CrossRef][Medline]
Bodenhausen, G. and Ruben, D.J. 1980. Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69: 185–189.[CrossRef]
Breneman, C.M. and Wiberg, K.B. 1990. Determining atom-centered monopoles from molecular electrostatic potential: The need for high sampling density in formamide conformational analysis. J. Comput. Chem. 11: 361–373.[CrossRef]
Cardinali, D.P., Golombek, D.A., Rosenstein, R.E., Cutrera, R.A., and Esquifino, A.I. 1997. Melatonin site and mechanism of action: Single or multiple? J. Pineal Res. 23: 32–39.[Medline]
Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham III, T.E., Ross, W.S., Simmerling, C.L., Darden, T.A., Merz, K.M., Stanton, R.V., Cheng, A.L., et al. 1999. AMBER 6. University of California, San Francisco.
Cook, W.J., Walter, L.J., and Walter, M.R. 1994. Drug binding by calmodulin: Crystal structure of a calmodulin–trifluoperazine complex. Biochemistry 33: 15259–15265.[CrossRef][Medline]
Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz Jr., K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., and Kollman, P.A. 1995. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117: 5179–5197.[CrossRef]
Craven, C.J., Whitehead, B., Jones, S.K., Thulin, E., Blackburn, G.M., and Waltho, J.P. 1996. Complexes formed between calmodulin and the antagonists J-8 and TFP in solution. Biochemistry 35: 10287–10299.[CrossRef][Medline]
Crivici, A. and Ikura, M. 1995. Molecular and structural basis of target recognition by calmodulin. Annu. Rev. Biophys. Biomol. Struct. 24: 85–116.[CrossRef][Medline]
Dai, J., Inscho, E.W., Yuan, L., and Hill, S.M. 2002. Modulation of intracellular calcium and calmodulin by melatonin in MCF-7 human breast cancer cells. J. Pineal Res. 32: 112–119.[CrossRef][Medline]
Delaglio, F., Grzesiek, S., Vuister, G., Zhu, G., Pfeifer, J., and Bax, A. 1995. NMRPipe: A multidimensional spectral processing system based on unix pipes. J. Biomol. NMR 6: 277–293.[Medline]
Essman, U., Perera, L., Berkowitz, M.L., Darden, T., Lee, H., and Pedersen, L.G. 1995. A smooth particle mesh Ewald method. J. Chem. Phys. 103: 8577–8593.[CrossRef]
Ford, J.M. and Hait, W.N. 1990. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 42: 155–199.[Medline]
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Zakrzewski, V.G., Montgomery Jr., J.A., Stratmann, R., Burant, J., et al. 1998. Gaussian 98, Rev. A7. Gaussian, Inc., Pittsburgh, PA.
Gilson, M.K., Given, J.S., Bush, B.L., and McCammon, J.A. 1997. The statistical-thermodynamic basis for computation of binding affinities: A critical review. Biophys. J. 72: 1047–1069.
Goodsell, D.S. and Olson, A.J. 1990. Automated docking of substrates to proteins by simulated annealing. Proteins 8: 195–202.[CrossRef][Medline]
Harmat, V., Bocskei, Z., Naray-Szabo, G., Bata, I., Csutor, A.S., Hermecz, I., Aranyi, P., Szabo, B., Liliom, K., Vertessy, B.G., et al. 2000. A new potent calmodulin antagonist with arylalkylamine structure: Crystallographic, spectroscopic and functional studies. J. Mol. Biol. 297: 747–755.[CrossRef][Medline]
Hickie, R.A., Wei, J.W., Blyth, L.M., Wong, D.Y., and Klaassen, D.J. 1983. Cations and calmodulin in normal and neoplastic cell growth regulation. Can. J. Biochem. Cell Biol. 61: 934–941.[Medline]
Hoeflich, K.P. and Ikura, M. 2002. Calmodulin in action: Diversity in target recognition and activation mechanisms. Cell 108: 739–742.[CrossRef][Medline]
Hori, K., Kushick, J.N., and Weinstein, H. 1988. Structural and energetic parameters of Ca2+ binding to peptides and proteins. Biopolymers 27: 1865–1886.[CrossRef][Medline]
Huerto-Delgadillo, L., Anton-Tay, F., and Benitez-King, G. 1994. Effects of melatonin on microtubule assembly depend on hormone concentration: Role of melatonin as a calmodulin antagonist. J. Pineal Res. 17: 55–62.[Medline]
Ikura, M., Kay, L.E., and Bax, A. 1990. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins: Heteronuclear triple-resonance three-dimensional NMR spectroscopy: Application to calmodulin. Biochemistry 29: 4659–4667.[CrossRef][Medline]
Jorgensen, W.L., Chandreskhar, J., Madura, J.D., Impey, R.W., and Klein, M.L. 1983. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79: 926–935.[CrossRef]
Jorgensen, W.L., Buckner, J.K., Boudon, S., and Tirado-Rives, J. 1988. Efficient computation of absolute free energies of binding by computer simulations. Application to the methane dimer in water. J. Chem. Phys. 89: 3742–3746.[CrossRef]
Kale, L., Skeel, R., Bhandarkar, M., Brunner, R., Gursoy, A., Krawetz, N., Phillips, J., Shinozaki, A., Varadarajan, K., and Schulten, K. 1999. NAMD2: Greater scalability for parallel molecular dynamics. J. Comp. Phys. 151: 283–312.[CrossRef]
Karasek, M., Reiter, R.J., Cardinali, D.P., and Pawlikowski, M. 2002. Future of melatonin as a therapeutic agent. Neuroendocrinol. Lett. 1: 118–121.
Kollman, P.A. 1993. Free energy calculations: Applications to chemical and biochemical problems. Chem. Rev. 93: 2395–2417.[CrossRef]
León, J., Macias, M., Escames, G., Camacho, E., Khaldy, H., Martin, M., Espinosa, A., Gallo, M.A., and Acuña-Castroviejo, D. 2000. Structure-related inhibition of calmodulin-dependent neuronal nitric-oxide synthase activity by melatonin and synthetic kynurenines. Mol. Pharmacol. 58: 967–975.
Liu, J., Qi, S., Zhu, H., Zhang, J., Li, Z., and Wang, T. 2002. The effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo. Chin. Med. J. (Engl.) 115: 759–762.[Medline]
Luo, H. and Sharp, K. 2002. On the calculation of absolute macromolecular binding free energies. Proc. Natl. Acad Sci. 99: 10399–10404.
MacNeil, S., Griffin, M., Cooke, A.M., Pettett, N.J., Dawson, R.A., Owen, R., and Blackburn, G.M. 1988. Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry. Biochem. Pharmacol. 37: 1717–1723.[CrossRef][Medline]
Marion, D., Driscoll, P.C., Kay, L.E., Wingfield, P.T., Bax, A., Gronenborn, A.M., and Clore, G.M. 1989. Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: Application to interleukin. Biochemistry 28: 6150–6156.[CrossRef][Medline]
Meador, W.E., Means, A.R., and Quiocho, F.A. 1992. Target enzyme recognition by calmodulin. 2.4 Å structure of a calmodulin–peptide complex. Science 257: 1251–1255.
Menéndez-Pelaez, A., Poeggeler, B., Reiter, R.J., Barlon-Walden, L.R., Pablos, M.I., and Tan, X. 1993. Nuclear localization of melatonin in different mammalian tissues. Immunochemical and radioimmunoassay evidence. J. Cell Biochem. 53: 373–382.[CrossRef][Medline]
Mori, S., Abeygunawardana, C., Johnson, M.O., and van Zijl, P.C. 1995. Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. J. Magn. Reson. B 108: 94–98.[CrossRef][Medline]
Morris, G.M., Goodsell, D.S., Halliday, R.S., Huey, R., Hart, W.E., Belew, R.K., and Olson, A.J. 1998. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J. Comp. Chem. 19: 1639–1662.
|
