The binding structure and affinity of photodamaged duplex DNA with members of the photolyase/cryptochrome family: A computational study

Abstract

Photolyases (PHRs) and cryptochromes (CRYs) belong to the same family known as blue-light photoreceptors. Although their amino acid sequences and corresponding structures are similar to each other, they exert different functions. PHRs function as an enzyme to repair UV-induced deoxyribonucleic acid (DNA) lesions such as a cyclobutane pyrimidine dimer (CPD) and a (6-4) photoproduct ((6-4)pp), whereas CRYs are a circadian photoreceptor in plants and animals and at the same time they control the photoperiodic induction of flowering in plants. When a new type cryptochrome was identified, it was assumed that another type of CRYs, cryptochrome-DASH (CRY-DASH), which is categorized as a subfamily of photolyase/cryptochrome family, would possess the DNA photolyase activity. However, CRY-DASH had a weak DNA photolyase activity, but the reason for this is still unclear. To clarify the reason, we performed molecular dynamics (MD) simulations for a complex of CPD-PHR or CRY-DASH with damaged double-stranded DNA (dsDNA) and estimated the binding free energy, ΔG_bind, between the protein and the damaged dsDNA by using a molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. ΔG_bind for both proteins were -35 and 57 kcal mol^-1, respectively, indicating that the structural stability of CRY-DASH was lower than that of CPD-PHR upon the damaged dsDNA binding. In particular, the number of amino acid residues relevant to the damaged dsDNA binding on the CRY-DASH surface was smaller than that on CPD-PHR. Therefore, the present result suggests that CRY-DASH has a weak DNA photolyase activity because it has a lower binding affinity than CPD-PHR.

Keywords: binding free energy; duplex DNA binding; molecular mechanics/Poisson-Boltzmann surface area; photolyase/cryptochrome family.

Figure 1

Conformational changes from normal thymine bases (TpT) to UV-lesions. (left) cyclobutane pyrimidine dimer (CPD). (right) (6-4) photoproduct. These conformations are induced by UV light absorption, and UV lesions are repaired by photolyases.

Figure 2

Repair mechanism of CPD by CPD-PHR.

Figure 3

Duplex DNA binding scheme for CPD-PHR and CRY-DASH. The base flipping of the lesion moiety will occur by the interaction between UV-damaged duplex DNA and protein. But due to the interaction of CRY-DASH is weak, the base flipping might not occur in CRY-DASH. Next, CPD-PHR and CRY-DASH recognize the lesion moiety and bind the duplex DNA. Here the binding affinity of CRY-DASH will be less than that of photolyase.

Figure 4

Computational schemes of the binding free energies based on MM/PBSA. The free energies colored in black are directly calculated, while the free energy of interest colored in red is indirectly did using the thermodynamic cycle of other free energies.

Figure 5

Definition of bond distance among amino acid residue, FADH⁻, and CPD. E282 (E326) and N348 (N392) are the amino acid residue in CPD-PHR (CRY-DASH). d₁, d₂, d₃, d₄, d₅, and d₆ are the distances between O1 of CPD and O1 of E282 (E326), between N2 of CPD and O2 of E282 (E326), between N1 of CPD and O of N348 (N392), between O2 of CPD and N of N348 (N392), between O1 of CPD and N of FADH⁻, and between O2 of CPD and N of FADH⁻, respectively

Figure 6

Active sites strucutres of CPD-PHR and CRY-DASH with the lowest RMSD values. Dotted lines correspond to the bond among amino acid residures, FADH⁻, and CPD. And dotted arrows represent the position the hydogen bond is formed.

Figure 7

Amino acid residues which have the partial binding free energy contribution lower than −1.0 kcal mol⁻¹. (a) The common amino acid residues in both CPD-PHR and CRY-DASH are Val147 (194), Tyr148 (195), Lys160 (205), Arg231 (277), Trp285 (329), Arg286 (330), Asn348 (392), Arg349 (393), Arg351 (395), Arg403 (447), Phe405 (449). Here, the numbers in parenthesis are the residue numbers in CRY-DASH. (b) The non-common amino acid residues in CPD-PHR are Arg10, Arg106, Ser139, Gly140, Tyr145, Gly149, Pro150, Asn154, Lys247, Arg273, Arg277, Arg305, Met352, Lys360, Arg367, Trp391, Pro399, Pro401, Asn406, Ser409, Gln410, Lys412, Lys413, Lys434, Arg459, Lys461, Lys464, and Lys471, and those in CRY-DASH are Thr196, Gln197, Arg199, Lys200, Arg259, Kys274, Lys288, Arg334, Lys339, Arg351, Arg404, Asn441, Pro443, Arg444, Lys453, Asn457, and Lys496.

Figure 8

Contact surface between protein and DNA. Blue and red color surfaces are duplex DNA with FADH⁻, and the amino acid residues that largely contribute to the DNA binding, respectively.