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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Stabilization and Crystallization of the Response Regulator NarL in its Phosphorylated State and in Complex with DNA

The response regulator NarL has been stably phosphorylated and bound to DNA for crystallization studies. The two-component Nar system in E. coli enables the organism to utilize nitrate and nitrite for respiration when oxygen is limited. Two membrane proteins detect the presence of nitrate or nitrite, autophosphorylate on a histidine residue, and then transfer the phosphate group to NarL. NarL-phosphate (NarLp) activates genes required for nitrate reductase and represses genes for other nonessential operons.

We are interested in determining the structure of NarLp alone and in complex with DNA to study protein conformation and DNA-recognition. Phosphorylation of NarL occurs on aspartate-59 in the N-terminal domain, while the C-terminal domain binds DNA. The crystal structure of NarL suggests that NarLp must undergo a conformation change between the two domains as a prerequisite for binding DNA. However, like many response regulators, NarLp is short-lived in vivo, which makes trapping the protein in its active state difficult. For this reason initial structural studies focused on the NarL C-terminus bound to DNA. Using acetyl phosphate as a phosphate donor, NarLp has been sustained for at least two weeks under high salt conditions and in the presence of certain crystallization additives.

NarLp can bind its DNA consensus sequence as a monomer or a dimer. Its various binding sites in the genome consist of direct or inverted repeats separated by two or three base pairs. Sequences containing these different arrangements have been designed and tested for NarLp binding. The constitutive mutant NarLS80R does not require phosphorylation and can bind DNA. Gel shift assays reveal that optimal sequences for NarLp and NarLS80R are DNA strands of 20 bases in length containing inverted, dimeric binding sites with a two base-pair spacing. Crystallization studies of these stable complexes are in progress.

Gail Katsir(1), Maria K-Grzeskowiak(2), Ann E. Maris(1), Mary L. Kopka(2), Robert P. Gunsalus(3), Mike Jarvis(3), Imke Schroder(3), and Richard E. Dickerson(1,2).

Chemistry and Biochemistry(1), Molecular Biology Institute(2), Microbiology and Molecular Genetics(3), UCLA, Los Angeles, CA 90095-1570
Phone: 310-206-8270, Fax: 310-267-1957, Email: katsir@mbi.ucla.edu