DNA-BINDING DOMAIN OF OmpR, A DIFFICULT STRUCTURE DETERMINATION. Erik Martínez-Hackert*, Helen Berman* and Ann Stock^&#, CABM, *Dept. of Chem., Rutgers. Univ., ^&Dept. of Biochem., UMDNJ & ^#HHMI

Escherichia coli respond to changes in osmotic pressure by altering the expression of two porin proteins, OmpF and OmpC, located in the outer membrane. The differential expression of the ompF and ompC genes is regulated by two proteins that belong to the ubiquitous two component family of regulatory proteins, the histidine kinase, EnvZ, and the response regulator, OmpR. Response regulator proteins are characterized by a conserved regulatory domain of approximately 130 amino acids and often contain a C-terminal effector domain. The N-terminal domain catalyzes transfer of a phosphoryl group from the histidine kinase. The activity of the C-terminal or downstream effector domain is controlled by the phosphorylation state of the regulatory domain. Response regulators are in many cases transcription factors with the C-terminal end comprising a DNA recognition domain.OmpR belongs to a family of at least 40 homologous response regulators with a DNA-binding domain of approximately 110 amino acids. This group of transcription factors does not have sequence homology with DNA-binding proteins of known three-dimensional structure, thus a novel DNA-binding motif might be expected. The structure determination of the C-terminal domain of OmpR, OmpRc, should define a protein fold and provide insight regarding DNA recognition and perhaps the mechanism of transcriptional regulation for a large family of transcription factors.

We have obtained two new crystal forms of an OmpR C-terminal fragment that includes amino acids 130-239. Crystals belong to the trigonal spacegroup P3_n12 with cell dimensions a=b=54.4 Å, c=130.5 Å and g=120.00. A second crystal form has been obtained by soaking this crystal form in a cryo-buffer and flash-cooling to 108 K in a liquid nitrogen stream. Crystals belong to the trigonal spacegroup P3_n12 with cell dimensions a=b=107.8 Å, c=130.5 Å and g=120.00. Both crystal forms diffract to at least 2.3 Å at a synchrotron light source.

We have encountered several difficulties during this structure determination: 1) An exhaustive search for heavy atom derivatives did not produce useful derivatives. 2) Cell axis c can vary more then 2%. 3) SeMet data was collected, but Se could not be positioned. We report a strategy to introduce single-site cysteine mutations for use in heavy atom derivatization. We have successfully collected one heavy atom dataset. Structure determination is in progress.