Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli.
Bottom Line: The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form.This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present.This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.
Affiliation: From the Department of Biochemistry, University of California, Riverside, California 92521.Show MeSH
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Mentions: To evaluate the significance of the bound cations and to resolve the long, disordered, polar region, we set out to crystallize a C-terminal fragment of BipA consisting of domains III and V and the C-terminal domain in the presence of magnesium ions (Fig. 2A). This fragment of BipA crystallized in the P41212 space group in the presence of 1 mm magnesium ions and diffracted to 2.5 Å. Based on the calculation of the Matthews coefficient, two molecules were expected in the asymmetric unit, but the native Patterson map and the self-rotation function did not reveal any non-crystallographic symmetry. Furthermore, molecular replacement with Molrep (28) and Phaser-MR (17) with different truncations of V. parahaemolyticus BipA (PDB ID: 3E3X) as search models found only reasonable solutions for one protomer in the asymmetric unit cell. The final structure was solved and refined as described under “Experimental Procedures” and converged to Rwork of 19.4% and Rfree of 22.7%.
Affiliation: From the Department of Biochemistry, University of California, Riverside, California 92521.