Ж У Р Н А Л С Т Р У К Т У Р Н О Й Х И М И И 2009. Том 50, 5 Сентябрь октябрь С. 873 877 UDK 539.27 STRUCTURAL STUDIES OF L-SERYL-L-HISTIDINE DIPEPTIDE BY MEANS OF MOLECULAR MODELING, DFT AND 1 H NMR SPECTROSCOPY 2009 Y. Liu 1, J.-B. Hou 1, X.-X. Liu 1, F.-M. Miao 2, Y.-F. Zhao 1,3 * 1 The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemistry, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, P.R. China 2 Chemical and Life College, Tianjin Normal University, Tianjin, 300074, P.R. China 3 The Key Laboratory for Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, P.R. China Received July, 13, 2008 L-Seryl-L-histidine dipeptide is of scientific interest mainly because of its various biological activities. In this paper, the lowest energy structure of the dipeptide was determined by molecular modeling, and further validated by quantum chemistry calculations and 1 H NMR spectroscopy. K e y w o r d s: Ser-His, dipeptide, molecular modeling, DFT, lowest energy structure. Cyclophilin A (CyPA) from human T lymphocyte consists of only 165 amino acid residues and exhibits peptidyl-prolyl cis-trans isomerase activity [ 1, 2 ]. As one of the most important members of CyP family, CyPA can catalyze protein's folding, assemblage and transportation, adjust the process of signal transduction, and participate in the immunosuppressive functions of Cyclosporin A (CsA) [ 2, 3 ]. The interaction of CyPA with dipeptides is attracting more and more attention of the researchers [ 4, 5 ]. It is well known that many small peptides possess various biological activities, especially L-seryl-L-histidine (Ser-His) [ 6 9 ]. It was discovered in our laboratory that Ser-His could bind to CyPA in the course of NMR titration. In our research project, theoretical calculation method has been used to explore the molecular mechanisms of the molecular interaction between CyPA and Ser-His. However, it was found that the molecular structure of Ser-His has not been reported yet. Considering the difficulty of obtaining a single crystal of Ser-His, alternatively, we determined the structure of Ser- His by molecular modeling, quantum chemistry calculations and 1 H NMR spectroscopy. CALCULATION METHOD AND EXPERIMENTAL DETAILS Molecular modeling studies were performed using molecular modeling software package SYBYL 7.1 [ 10 ] on SGI workstation with IRIS operating system. Quantum chemistry ab initio calculations were performed on a PC cluster with 20 CPU and at B3LYP/6-311G** level using the Gaussian-03 software [ 11 ]. 1 H NMR spectrum was recorded on Bruker AV 400 MHz NMR spectrometers with D 2 O and calibrated internally using TMS as a reference. Ser-His was purchased as an acetate salt from Bachem (Switzerland, HPLC purified). * E-mail: yfzhao@xmu.edu.cn
874 Y. LIU, J.-B. HOU, X.-X. LIU ET AL. Fig. 1 (left). Structure and atom numbering scheme of Ser-His dipeptide Fig. 2 (right). Eight representative conformations of Ser-His: the unity of energy is kcal/mol, H-bonds are shown by dashes Fig. 3 (below). The global minimum conformation of Ser-His obtained by molecular mechanics method RESULTS AND DISCUSSION Modeling the structure of Ser-His by molecular mechanics. The structure and atom numbering scheme of Ser-His is shown in Fig. 1. From the Figure, it was found that there are nine rotatable single bonds in the molecule, excluding those involving hydrogen atoms. Hence, conformational searches were performed on Ser-His using random search method, with Tripos molecular mechanical parameters, and the MAX-HITS number set to 5. Energy cutoff value was set to 70 kcal/mol. Then, a conformational set containing 538 conformers was obtained. When the value of RMSD was set to 1.2 Å as a criterion to distinguish different groups, eight representative conformations of Ser-His were observed through conformational analysis, as illustrated in Fig. 2. The conformational analysis shows that the typical conformations Ser-His can adopt are either folded or extended shapes, implying that Ser-His is very flexible. For example, in Fig. 2, the conformation of the molecule M1 is a folded shape, and the conformation of the molecule M6 is extended shape. The other conformations of the molecules are between the two shapes. The flexibility of the dipeptide gives it the ability to complex with a wide range of substrates that possess complementary structures. The folded conformation possesses the lowest energy. This information is useful for our study on the interaction of Ser-His with CyPA by molecular modeling method in the future.
STRUCTURAL STUDIES OF L-SERYL-L-HISTIDINE DIPEPTIDE 875 Fig. 4. Energy fluctuation plot of the modeled structure of Ser-His Fig. 5. The fluctuation plot of distance between carbon atom C2 and C21 in the modeled structure of Ser-His In the conformational set, the global minimum conformation is shown in Fig. 3, with reliability of above 96 %. To validate the stability of the global minimum conformation obtained, molecular dynamics simulation has been taken with parameters: 30 ps, time step 1 fs, sampling interval 5 fs, in canonical ensemble at 25 C. Obtained from the data energy fluctuation of the molecule is shown schematically in Fig. 4. Energy fluctuation from the average value is about 2 kcal/mol after the equilibrium reached; the fluctuation of distance between -carbon atom C2 and C21 is schematically illustrated in Fig. 5. Distance fluctuation from the average value is about 0.13 Å after the equilibrium reached. The above experiment results indicate that the modeled structure of Ser-His is stable. Ab initio calculations of the molecular structure of Ser-His. In order to prove the structure of Ser-His obtained by molecular mechanics method further, quantum chemistry ab initio calculations were performed. The structure of Ser-His (Fig. 3) was optimized with DFT B3LYP method and 6-311G** base set using the Gaussian03 software. After 18 cycles, the convergence level was 0.5009D-08, and the Hiessen matrix analysis indicated that no imaginary frequency was found for the optimized structure, as shown in Fig. 6. Based on the above calculation, the optimized structure is stable and credible; the atomic coordinates are summarized in Table 1. The numerical values of bond distances, bond angles and dihedral angles, obtained by ab initio calculations, were consistent with those obtained by the molecular mechanics modeling method. The corresponding dihedral angle values obtained by the two calculation methods are summarized in Table 2. The result of molecular fit calculation showed that the value of RMSD is equal to 0.188 Å. The sketch map of molecular fit is shown in Fig. 7. Therefore, the above calculation results proved further that the molecular structure of Ser-His obtained by molecular modeling method is credible. Fig. 6. The global minimum conformation of Ser-His Fig. 7. The superposition plot of the global minimum
876 Y. LIU, J.-B. HOU, X.-X. LIU ET AL. obtained by quantum chemistry ab initio method The atom coordinates of the optimized structure of Ser-His conformation of Ser-His obtained by the two calculation methods T a b l e 1 Atom Coordinates (Å) X Y Z Atom Coordinates (Å) X Y Z N1 0.022115 0.010697 0.012880 H17 3.963422 0.384311 0.434875 C2 0.006438 0.010541 1.451965 H18 3.100732 3.656575 0.238363 C3 1.439011 0.008092 2.057492 C19 0.675670 0.856250 0.814112 C4 0.701980 1.377913 1.809160 O20 1.212393 1.913293 0.523479 O5 1.069374 1.523411 2.966625 C21 0.720782 0.473374 2.315832 C6 2.370645 0.882144 1.278427 C22 2.169138 0.241886 2.823992 N7 2.146426 2.225472 1.036478 O23 2.511673 1.122538 2.649272 C8 3.468728 0.571320 0.514650 N24 0.024505 0.815415 2.626346 C9 3.071460 2.645350 0.138242 H25 0.359217 0.849852 3.589951 N10 3.895259 1.670843 0.208162 H26 0.239378 1.284871 2.861354 O11 0.716729 2.208859 0.831083 H27 2.822153 0.923941 2.279157 H12 0.210608 0.981773 0.235593 H28 2.229122 0.472877 3.892212 H13 0.568370 0.820655 1.859558 H29 3.147265 1.253803 1.907464 H14 1.337965 0.330042 3.097833 H30 0.812640 0.972074 1.991629 H15 1.847942 1.003496 2.052460 H31 0.701732 1.553893 2.504568 H16 1.279603 2.708914 1.265637 T a b l e 2 The dihedral angles (deg.) in main chain of the molecular structure obtained by the two calucation methods Dihedral Angles Molecular Mechanics Method Quantum Chemistry Ab Initio Method Dihedral Angles Molecular Mechanics Method Quantum Chemistry Ab Initio Method D(C2,N1,C19,C21) 177.5 168.8 D(C2,N1,C19,O20) 2.3 13.4 D(C19,N1,C2,C4) 162.1 169.6 D(N1,C2,C4,O5) 156.7 166.4 1 H NMR chemical shift (ppm) of Ser-His obtained in experiment and theoretical calculations T a b l e 3 1 H NMR Chemical Shift Experimental Results* (D 2 O) Theoretical Calculation Results (exp. cal.) 1 H NMR Chemical Shift Experimental Results* (D 2 O) Theoretical Calculation Results (exp. cal.) 16-H 10.3925 27-H 4.018 4.2327 0.2147 12-H 9.8226 15-H 3.115 3.5476 0.4326 31-H 7.7036 28-H 4.018 3.3465 0.6715 18-H 8.451 7.2505 1.2005 13-H 4.406 3.3336 1.0724 17-H 7.163 6.7098 0.4532 26-H 4.012 3.0709 0.9411 29-H 4.5412 14-H 3.115 3.0547 0.0603 30-H 4.3703 25-H 2.9802 * The active hydrogen was not determined in D 2 O by 1 H NMR.
STRUCTURAL STUDIES OF L-SERYL-L-HISTIDINE DIPEPTIDE 877 The 1 H NMR chemical shift measurement on Ser-His. In order to validate the molecular conformation of Ser-His obtained by theoretical calculation method, 1 H NMR experiment was performed in D 2 O at 25 C, with TMS as a reference. At the same time, 1 H NMR spectrum was calculated at DFT B3LYP/6-311G** level using the Gaussian-03 software, on the global minimum conformation obtained by quantum chemistry ab initio method (Fig. 6). 1 H NMR chemical shifts for Ser-His obtained in experiment and theoretical calculations are summarized in Table 3. From the comparison with the chemical shift values in Table 3, it was observed that the minimum difference is 0.060 ppm, the maximal difference is 1.200 ppm, and average difference is 0.469 ppm, with RMS = 0.74 ppm. Therefore, the NMR results obtained by theoretical calculations are mainly consistent with the NMR experiment results. CONCLUSION From molecular modeling, quantum chemistry calculations and 1 H NMR spectroscopy, the molecular structure of Ser-His dipeptide was determined. The theoretical calculation work of this paper is of high significance for our studies exploring the mechanisms of molecular interaction between CyPA and Ser-His by molecular modeling. Acknowledgements. This work was supported by the Chinese National Science Foundation (grant N 20732004) and the Research Fund for the Doctoral Program of Higher Education of China (grant N 200803841009). REFERENCES 1. Handschumacher R.E., Harding M.W., Rice J. et al. // Science. 1984. 226, N 4674. P. 544 547. 2. Fischer G., Wittmann-Liebold B., Lang K. et al. // Nature. 1989. 337, N 6206. P. 476 478. 3. Takahashi N., Hayano T., Suzuki M. // Nature. 1989. 337, N 6206. P. 473 475. 4. Ke H.M., Mayrose D., Cao W. // Proc. Natl. Acad. Sci. U.S.A. 1993. 90, N 8. P. 3324 3328. 5. Zhao Y.D., Ke H.M. // Biochem. 1996. 35, N 32. P. 7362 7368. 6. Li Y.S., Zhao Y.F., Hatfield S. et al. // Bioorg. Med. Chem. 2000. 8, N 12. P. 2675 2680. 7. Chen J., Wan R., Liu H. et al. // Chem. J. Chinese Universities. 2001. 22, N 8. P. 1349 1351. 8. Chen J., Wan R., Liu H. et al. // Lett. Pept. Sci. 2000. 7. P. 325 329. 9. Du H.L., Wang Y.T., Yang L.F. et al. // Ibid. 2002. 9. P. 5 10. 10. SYBYL, version 7.1, Tripos Associates: St. Louis, MO, 1998. 11. Frisch M.J., Trucks G.W., Schlegel H.B. et al. Gaussian-03, Revision C.02. Gaussian, Inc., Wallingford CT, 2004.