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33ISSN 2072-5981
Volume 14, 2012 No. 2, 12202 - 5 pages
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”Magnetic Resonance in Solids. Electronic Journal” (MRSe/) is a
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Editors-in-Chief Jean Jeener (Universite Libre de Bruxelles, Brussels) Boris Kochelaev (KFU, Kazan) Raymond Orbach (University of California, Riverside)
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Vadim Atsarkin (Institute of Radio Engineering and Electronics, Moscow) Detlef Brinkmann (University of Zurich, Zurich) Yurij Bunkov (CNRS, Grenoble) John Drumheller (Montana State University, Bozeman) Mikhail Eremin (KFU, Kazan) Yoshio Kitaoka (Osaka University,
Osaka)
Boris Malkin (KFU, Kazan) Haruhiko Suzuki (Kanazawa University, Kanazava) Murat Tagirov (KFU, Kazan)
*
In Kazan University the Electron Paramagnetic Resonance (EPR) was discovered by Zavoisky E.K. in 1944.
NMR studies and molecular dynamics simulation of cyclosporin in complex
with detergent micelles1
S.V. Efimov *, V.V. Klochkov Kazan Federal University, Kremlevskaya, 18, Kazan 420008, Russia *E-mail: [email protected] (Received November 19, 2012; accepted November 30, 2012)
Cyclosporin A is a highly hydrophobic peptide, but its complex with sodium dodecyl sulphate micelles can be readily dissolved in water. Nuclear magnetic resonance (NMR) investigations of cyclosporin bound to detergent micelles were carried out (including NOE spectroscopy) and yielded internuclear distances for a set of atom pairs. Based on these structural data, conformation of cyclosporin was obtained by means of molecular dynamics simulation.
PACS: 82.56.-b, 82.70.Uv, 87.14.ef, 87.15.B-.
Keywords: peptide conformation, model membrane, NOE spectroscopy, molecular dynamics simulation
1. Introduction
Cyclosporin A (CsA), cyclo(-Bmt-Abu-Sar-Mle-Val-Mle-Ala-DAla-Mle-Mle-Mva-), is a peptide used in therapeutics as an immunosuppressant. It acts through formation of a complex with the 18-kDa protein cyclophilin (Cyp18). This protein is ubiquitous, and the CsA-Cyp18 complex accumulates in cytoplasm in many types of cells; however, cyclosporin influences selectively T-cells. Apparently, cell recognition occurs, involving receptor proteins on cell membranes [1].
The task of determination of the structure of a molecule bound to a cell membrane is thus of a great importance. Phospholipid vesicles are the best structures representing cell membranes in vitro; however, NMR-based structure determination for molecules bound to heavy vesicles is hampered due to short transverse relaxation times. Fortunately, useful information can be obtained if relatively small supramolecular structures, detergent micelles, are employed [2, 3].
Stable conformations of cyclosporin, which exist in apolar solvents, were studied by means of NMR and different computational approaches [4, 5]. More recently, the question of interaction of CsA and metal ions within detergent micelles was studied [6]. Structure of this peptide is found to be flexible and strongly dependent on the environment (solvent polarity, presence of ions, complex formation with larger molecules [7-9]). This fact calls forth wide experimental study of CsA.
2. Experimental
Cyclosporin A ( > 98.5%) was purchased from Sigma-Aldrich, Inc. and used without further purification. Sodium dodecyl sulphate in concentration exceeding the critical micelle concentration was used as the model-mimicking compound. Spectra were recorded on a Bruker Avance II spectrometer operating at the proton frequency of 500 MHz; heteronuclear measurements and some of the NOESY experiments were performed on a Bruker Avance at 700 MHz (Leipzig University). For the sake of signal assignment, heteronuclear correlation spectra were acquired: HSQC, HMBC, and band-selective HMBC. The latter excited carbonyl resonances in a relatively narrow band (3522 Hz, corresponding to 20 ppm in the 13C dimension). Geometric restraints, extracted from the NMR data,
f This paper material was selected at XV International Youth Scientific School "Actual problems of magnetic resonance and its application", Kazan, 22 - 26 October 2012. The paper was recommended to publication in our journal and it is published after additional MRSej reviewing.
NMR studies and molecular dynamics simulation of cyclosporin in complex with detergent micelles
were then employed as input data in molecular dynamics (MD) simulation. Ensembles of structures were built in the XPLOR-NIH 2.29 programme [10]. One hundred structures were generated in the simulated annealing protocol (6000 iterations at the initial temperature of 800 K, then cooling during 14000 iterations down to 100 K).
3. Results and Discussion
One-dimensional 1H NMR spectrum of cyclosporin is shown in figure 1. Evidently, four resonances are dominant in the amide region (6.5-8.5 ppm); seven lines of NCH3-groups are notable in the region 2.7-3.6 ppm. Signals of amide protons are absent in the spectrum recorded in D2O due to intermodular exchange with deuterium; signals of a-protons are suppressed in the H2O/D2O sample together with the solvent resonance. One conformation is thus dominant, and we can assign the signals using two-dimensional correlation experiments TOCSY, (13C,1H)-HSQC, (13C,1H)-HMBC, and NOESY.
Increase in temperature causes small changes in the amide resonances. The signal of Abu2 shifts significantly to the higher field (0.018 ppm/K in the range from 10 to 35°C), what is interpreted usually as an evidence of outward NH bond, preventing the formation of an intramolecular hydrogen bond [11]. Alanine NH signal has the temperature gradient of 0.005; the other two protons (D-Ala8 and Val5) show A5/AT < 0.003 ppm/K, which may be an evidence of intramolecular hydrogen bonds involving the latter two protons. The minor signals also move to the higher field in a similar manner. Their intensity remains the same.
Some information on the spatial structure of a molecule is contained in chemical shifts. Systematic deviations in signals of backbone protons to the higher or lower field mean that corresponding backbone region is a part of an a- or p-structure, respectively [12]. On a qualitative level, comparison of 1H and 13C spectra can give an evidence of similarity or dissimilarity of molecular conformations in different solvents. Proton spectra are sensitive to local environments (including solvent effects), while 13C resonances depend mainly on local conformations. In general, 13C NMR spectra of CsA in apolar solvents and in micellar solution are similar (fig. 2). The sequence of the Ca signals in all three media is this: residue 1 (formally also 12) is the leftmost, then we observe 11 and 10; closely located three signals 4, 5, and 6; after an empty interval of ~5 ppm stand signals of residues 3, 2, 7, 9, and 8. Proton spectra, despite their narrow frequency range and relatively big variations in chemical shifts, can also be identified. For instance, the a-proton of Mle9 resonates always in the lowest field, while that of Ala7, in the highest field. We can thus expect that local conformations of each amino acid residue undergo subtle changes when going from one apolar medium to another. The same kind of information is provided by solid-state NMR spectra. In particular, similarity of 13C NMR spectra recorded in solid-
Figure 1. 1H NMR spectra of cyclosporin at 20°C. From top: the CsA-SDS micelles system, spectrum with water suppression; micellar solution based on D2O; chloroform. Solvent signals are marked by "S".
state and in solution proves existence of a single stable conformation in the liquid state [13]. In our case, comparison of the spectra seems difficult due to a large number of lines; nevertheless, arrangements of the peaks in organic solvents and in crystal are similar (fig. 3).
60 50 40 30 20 ppm
Figure 2. Comparison of 13C spectra of CsA in three media: (a) complex with SDS micelles (projection from an HSQC spectrum); (b) chloroform; (c) benzene.
150 100 50 PPm
Figure 3. Comparison of 13C spectra of CsA in three media: (a and b) CP-MAS recorded with different rotation speeds; 13C{1H} spectra in benzene (c) and chloroform (d).
NMR studies and molecular dynamics simulation of cyclosporin in complex with detergent micelles
Two-dimensional NOE spectroscopy provides information for constructing a model structure of cyclosporin. Analysis of NOESY spectra was made following [14-16]. Intensity of a cross-peak of the nuclei a and b is normalized by intensities of corresponding diagonal peaks (standing in the crosssection with the same indirect frequency F1). After that, dependencies of intensities on the mixing time I(Fu, F2b)/1(Fla, Fla) = f (Tm) are built. Atom pair Ala7(Ha-H3P) was chosen for calibration. Corresponding distance is 2.8 A, according to the jump model of averaging [17]; simpler averaging models yield lesser values (by 0.1-0.2 A). For the NCH3-groups, averaging R = {R- )-1/3 was used (possible variants are described in [18]).
One hundred structures were generated in the simulated annealing protocol; ten of them, having minimal energies, were selected. They are moderately close to each other; RMSDs between the lowest-energy structure and remaining nine molecules in positions of backbone atoms lie from 3.1 to 4.8 A. Some violations were observed: Mle10(Ha)-Mva(Hp), Mle10(NCH3)-Mle9(Ha) (after averaging the distances to the methyl protons), Mle6(NCH3)-Ala(Ha), and Sar(NCH3)-Bmt(Ha) were all longer that the NOE-based values. Analysis of NOEs underestimates the distances due to spin diffusion, when the coherence is transferred between atoms a and b via one or several intermediate atoms; the apparent cross-relaxation rate has no clear correlation with the distance rab in this case.
We repeated the simulation, having omitted the incorrect distances. The new ten structures with minimal energies showed backbone RMSDs from 2.6 to 4.6 A.
Figure 4 presents one of them (with the lowest energy) compared with the structure of cyclosporin in chloroform, modelled allowing for hydrogen bonds. This simulation was performed as described in [19]; information on hydrogen bonds was taken from literature data [7].
4. Conclusions
Several conformers of cyclosporin coexist in micellar solution based in SDS. Structure of the main conformer was determined with the aid of the NOESY method. The peptide ring is nearly planar; some differences compared to its structure in apolar organic solvents were found. Close contacts of Bmt Ha with a-protons of Mle6 and Ala7 are absent. The ring of cyclosporin becomes less oblate; chain regions with residues 9-11, 1 and 4-6, which in chloroform are close to each other and resemble P-structure, move off from each other in the complex. This change may be due to steric interaction with SDS molecules which prevents formation of intramolecular hydrogen bonds. Note also that Ha chemical shifts in the micellar solution are, in general, smaller that in organic solvents. It gives an evidence of additional shielding of the peptide by surrounding detergent molecules.
Acknowledgements
Certain part of the work was performed in Leipzig University under the auspices of the "Evgenii Zavoiskii" grant of the Ministry of Science and Education of the Republic of Tatarstan and Deutscher Akademischer Austauschdienst.
Figure 4. Comparison of the CsA structures based on NOE data obtained in chloroform and in complex with SDS micelles. Illustration was prepared in the Chimera programme [20].
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