View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 2194 Biophysical Journal Volume 94 March 2008 2194–2203 Discrimination of Class I Cyclobutane Pyrimidine Dimer Photolyase from Blue Light Photoreceptors by Single Methionine Residue Yuji Miyazawa,* Hirotaka Nishioka,y Kei Yura,z§ and Takahisa Yamato*{ *Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; yGraduate School of Environmental and Human Science, Meijo University, Nagoya 468-8502, Japan; zQuantum Bioinformatics Team, Center for Computational Science and Engineering, Japan Atomic Energy Agency, Kyoto 619-0215, Japan; §Research Unit for Quantum Beam Life Science Initiative, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Kyoto 619-0215, Japan; and {CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan ABSTRACT DNA photolyase recognizes ultraviolet-damaged DNA and breaks improperly formed covalent bonds within the cyclobutane pyrimidine dimer by a light-activated electron transfer reaction between the flavin adenine dinucleotide, the electron donor, and cyclobutane pyrimidine dimer, the electron acceptor. Theoretical analysis of the electron-tunneling pathways of the DNA photolyase derived from Anacystis nidulans can reveal the active role of the protein environment in the electron transfer reaction. Here, we report the unexpectedly important role of the single methionine residue, Met-353, where busy trafficking of electron-tunneling currents is observed. The amino acid conservation pattern of Met-353 in the homologous sequences perfectly correlates with experimentally verified annotation as photolyases. The bioinformatics sequence analysis also suggests that the residue plays a pivotal role in biological function. Consistent findings from different disciplines of computational biology strongly suggest the pivotal role of Met-353 in the biological function of DNA photolyase. INTRODUCTION Ultraviolet (UV) light creates cyclobutane pyrimidine dimers Various methods for computation of the electronic factor (CPDs) in a DNA chain and causes skin cancer. DNA pho- in electron transfer reactions have been presented (9–11). tolyase, containing a flavin cofactor, absorbs light and repairs Three major computational methods have been developed for the UV-damaged DNA using the electron transfer reaction the long-range electron transfer reactions in biological sys- between the electron donor (flavin adenine dinucleotide, tems, namely, the extended Hu¨ckel approach used by Marcus FADHÀ) and the electron acceptor (CPD) (1). Improperly and Stuchebrukhov (12) and expanded by Kakitani and co- formed covalent bonds in CPD are broken as a result of the workers (13–15), the pathway approach by Beratan and electron transfer reaction, which is completed within 170 ps. Onuchic (16), the square barrier model of Hopfield (17) To analyze the spatial pattern of electron-tunneling path- promoted by Dutton (18), and the more intensive self-con- ways, Medvedev and Stuchebrukhov proposed an electron- sistent field molecular orbital methods (19,20). In this study, tunneling current and studied the electron transfer reaction of we employed the extended Hu¨ckel approach to analyze the the computationally predicted complex structure of Escherichia thermal fluctuation of electron-tunneling pathways for a large coli DNA photolyase and CPD (2). They proposed a scheme number of protein structures extracted from a molecular of indirect electron transfer via the adenine of FADHÀ as a dynamics simulation trajectory. mediator of electron tunneling from the donor to the acceptor It is widely accepted that the electronic factor for the in their pioneering work on the CPD photolyase complex electron transfer reaction fluctuates significantly along with model. In 2004, Mees et al. reported the complex structure of the thermal fluctuation of the protein environment (15,21– the DNA photolyase derived from Anacystis nidulans and the 24). Troisi et al. have made important contributions to the CPD analog by x-ray crystallography (3). The isoalloxazine analysis of the effects of thermal fluctuations on the donor- ring of the FADHÀ in the crystal structure is located close to acceptor couplings (25,26). Skourtis et al. have explicitly the CPD, indicating the possibility of direct electron transfer analyzed how thermal fluctuations affect the protein electron from the FADHÀ to the CPD (4,5), and recent theoretical transfer (27). Recently, Nishioka et al. proposed a non- analyses support the direct transfer (6). An active role of the Condon theory for the electron transfer of thermally fluctu- protein environment in electron transfer from the FADHÀ*to ating protein media (28), and another study has analyzed the the CPD has not been supported (1,7,8), but the possibility electron-tunneling pathways in a fixed protein environment of protein-mediated electron transfer is not completely ex- (2). In this study, we used molecular dynamics simulation cluded. and quantum mechanical calculations to study the electron transfer reaction from the excited flavin cofactor to the thy- mine dimer in the explicit protein environment of a thermally Submitted August 7, 2007, and accepted for publication November 5, 2007. fluctuating CPD photolyase complex (3). These calculations Address reprint requests to Takahisa Yamato, E-mail: yamato@phys. turned out to be helpful in identifying residues that are im- nagoya-u.ac.jp. portant in the electron transfer reaction. Furthermore, we Editor: David P. Millar. Ó 2008 by the Biophysical Society 0006-3495/08/03/2194/10 $2.00 doi: 10.1529/biophysj.107.119248 Theoretical Study on DNA Photolyase 2195 used sequence database search techniques to evaluate the ¼ + ; ¼ 1 ð i f À f i Þð À Þ; biological conservation of the key residues found by the Jab Jmn Jmn - CmCn CmCn Hmn ESmn (2) m2a;n2b h analysis. As a result of the combined use of multiple disci- i i where Cm; Cn are the coefficients of atomic orbitals fm; fn in Ci; and plines of computational biology, we discovered that the f f Cm; Cnare the coefficients of atomic orbitals fm; fn in Cf . The total electron methionine residue located at site 353 of A. nidulans CPD population should be conserved throughout the reaction from the initial state photolyase is a likely determinant for the function of class I i to the final state. To impose this conservationpffiffiffiffiffiffiffiffiffi condition,pffiffiffiffiffiffiffiffiffiffi we scale the Cm f i f i CPD photolyase in the blue light photoreceptor superfamily. inCi (Cmin Cf ) by multiplying 1/ Pop (1/ Pop ), where Pop ¼ + i i ; f ¼ + f f n;m2all atomic orbital CnSnmCm Pop n;m2all atomic orbital CnSnmCm. To find the important region for long-range intramolecular electron tunneling, MATERIALS AND METHODS analysis of the interatomic-tunneling current Jab is helpful. However, since the value of T fluctuates as the thermal fluctuation of the protein, direct Molecular dynamics simulations and electronic DA comparison of Jab for different protein structures is not useful. To compare structure calculations the significance of Jab among different protein structures, we introduce the normalized tunneling current K as K ¼ h- J =T . In the mediator region, The initial conformation of the complex of CPD analog and the DNA pho- ab ab ab DA the summation of the normalized tunneling current is always zero for a given tolyase of A. nidulans was extracted from the Protein Data Bank (PDB code atom, a, because the annihilation and creation of electrons do not take place. 1TEZ) (3). Crystallographic water molecules within the DNA photolyase and Therefore, the significance of electron tunneling cannot be evaluated for between the DNA photolyase and the CPD fragment were incorporated into each atom with the normalized tunneling current. To evaluate the amount of the system. This system was then immersed in an octahedral box of water the electron-tunneling current traffic, we introduce the electron-tunneling molecules. The total number of water molecules was 25,268. The AMBER99 count (35) as N ¼ h- +9 J =jT j; where the summation +9 is taken over force field (29) and the TIP3P model (30) were employed for the polypeptide a b ab DA À all positive tunneling currents J . The electronic states of the donor, accep- chain and water molecules, respectively. The force field for the FADH , ab tor, protein, and the crystallographic waters are solved at the extended CPD analog, and 8-hydroxy-5-deazaflavin (8-HDF) were developed based Hu¨ckel level. The extended Hu¨ckel parameters were taken from the litera- on FADH(À), thymine dimer, and flavin mononucleotide (FMN) in the ture (36), which are similar to those adopted by Stuchebrukhov et al. (37) in AMBER Parameter Database (http://pharmacy.man.ac.uk/amber), respec- their previous work (2). We confirmed that the extended Hu¨ckel calcula- tively. The atomic partial charges for these cofactors were calculated with the tion with this parameterization reproduced a result similar to that of the RESP scheme using the GAMESS (31) and AMBER8 (32) program pack- previous work (2) for the fixed environment of the protein medium directly ages. The 4-31G basis set was employed for this purpose. The bond lengths derived from x-ray crystallography (3). The value of T is calculated by of the equilibrium conformation were adopted from the x-ray crystallo- DA the pseudo-Green function technique (14,38) for each MD snapshot. The graphic values. The spring constants for the bond stretching and angles and electron-tunneling pathways are analyzed by drawing the map of interatomic- dihedral torsional barriers were obtained from the AMBER parameter da- tunneling currents (24). tabase. To construct the force field of the CPD analog, the structure of CPD To obtain the electron-tunneling pathways of CPD photolyase, we cal- was extracted from the PDB entry, 1SNH (33), and the ÀO–P –O– part was 2 culated electron-tunneling matrix elements and the interatomic electron- replaced with ÀO–CH –O–. Structure optimization of the system was per- 2 tunneling current for each of the instantaneous structures derived from the formed using the AMBER8 program with the harmonic restraints imposed molecular dynamics simulation.