DeoR is involved in B. subtilis carbon catabolism as the local repressor for transcription of enzymes digesting deoxyribose and deoxyribonucleosides. This repressor binds an operator DNA by its N‑terminal DNA‑binding domain (DBD) and blocks transcription of catabolic enzymes in the absence of an effector molecule. If the effector is present it binds to the C‑terminal effector‑binding domain (EBD) of the repressor and causes its release from the DNA binding site. The structures of the C‑terminal EBD of DeoR in free form and in the complex with the effector deoxyribose‑5’‑phosphate have already been reported [1]. Nevertheless, for the understanding of the allosteric effects of the repressor during the metabolic regulatory process, it is necessary to know the 3D structure of the full‑length protein in complex with its operator DNA. To achieve this, we initiated structural studies DeoR in complex with DNA operator.
Recombinant DeoR was prepared by heterologous expression in E. coli BL21 (DE3) and purified with yield of 3.4 mg per 1 L of bacterial culture. Crystallization of DeoR in complex with 18-base pair DNA duplex derived from the operator sequence was carried out using the vapor diffusion sitting‑ and hanging‑drop techniques. Needle‑shaped crystals were obtained and diffraction data were collected to maximal resolution of 3.6 Å Crystal structure was determined by molecular replacement and confirmed the presence of DeoR-DNA complex. At this resolution, however, DBD and DNA could not be unambiguously modelled into the electron density maps. Optimization of crystal quality to achieve higher resolution failed. As an alternative, we chose to perform structural study on DeoR DBD in complex with DNA to be used as a model for low-resolution structure.
DeoR DBD was prepared by heterologous expression in E. coli BL21 and purified with yield of 5.8 mg per 1 L of bacterial culture. Crystallization of DBD in complex with 15-base pair DNA duplex yielded crystals diffracting to 2.3 Å resolution. Crystal structure of DBD-DNA complex was solved by molecular replacement using the preliminary model from full-length DeoR structure. Refinement of crystallographic model is currently in progress.
1. J. Škerlová, M. Fábry, M. Hubálek, Z. Otwinowski, P. Řezáčová, FEBS J., 281 (2014), pp. 4280-4292.
This project was supported by the Ministry Education of the Czech Republic (programme “NPU I”) project LO1034.