W0036

Crystal Structure of the NarL Effector Domain-DNA Complex. Ann E. Maris(1), Michael Sawaya(2), Maria Grzeskowiak(2), Mike Jarvis(3), Mary L. Kopka(2), Robert Gunsalus(3), Richard E. Dickerson(1, 2), (1)Biochemistry & Molecular Biology, (2)Molecular Biology Institute, (3)Microbiology & Molecular Genetics, University of California, Los Angeles, CA 90095, maris@mbi.ucla.edu

The crystal structure of the NarL C-terminal domain (NarLc) in complex with DNA has been solved to 2.1 Å resolution. NarL is a two-domain response regulator of E. coli, whose DNA binding is regulated through two-component signal transduction. Once the CheY-like receiver domain of NarL receives the phosphorylation signal, conformational changes occur between the two domains, allowing NarL to bind DNA. NarL functions in vivo as an activator and a repressor at multiple operons encoding genes involved in anaerobic respiration. This is the first crystal structure of a two-component response regulator in complex with DNA.

The heptameric DNA sites recognized by NarL are found in both tandem and antiparallel arrangements; however, binding at the antiparallel sites appears cooperative with resultant higher affinity. In this first structure, the helix-turn-helix NarLc protein binds as a dimer to a 20mer oligonucleotide containing two antiparallel recognition sites. The two NarLc proteins form a dimerization interface at a region formerly involved in interdomain contact in the unactivated full-length protein, allowing us to model the conformational changes upon phosphorylation that must occur to allow DNA binding in the antiparallel arrangement.

Contacts in adjacent DNA major grooves cause DNA curvature of approximately 42 degrees. This major curvature occurs over the entire DNA helix, distinct from the large kinks which cause bending in other structures such the Cap-DNA complex. This continuous curvature is also found in eukaryotic transcription factors such as the homeodomain proteins, and probably provides an additional level of transcription regulation. Factors contributing to NarLc-DNA recognition are protein oligomerization, protein-DNA contacts, and the inherent deformability of particular DNA sequences.