Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli.
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.
- Enteropathogenic Escherichia coli/enzymology/genetics*/pathogenicity*
- Escherichia coli Proteins/chemistry*/genetics/metabolism
- GTP Phosphohydrolases/chemistry*/genetics/metabolism
- Guanosine Diphosphate/chemistry*/metabolism
- Crystallography, X-Ray
- Gene Expression
- Models, Molecular
- Protein Binding
- Protein Structure, Tertiary
- Recombinant Proteins/chemistry/genetics/metabolism
- Ribosome Subunits, Small/genetics/metabolism
- Signal Transduction
- Stress, Physiological
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Figure 1: Structure of full-length BipA in apo, GDP-, and ppGpp-bound forms.A, structures of BipA, LepA (PDB ID: 3CB4), and EF-G (PDB ID: 1FNM) with common domains I, II, III, and V displayed in red, yellow, pink, and blue, respectively. The EF-G specific domains, G′ and IV, are in orange and green, respectively. The LepA and the BipA specific C-terminal domains are in purple. The cobalt cation ions and their coordinated amine molecules are displayed as pink and blue spheres, respectively. Disordered regions are indicated with dashed lines. B, sequence alignment of EF-G, LepA, BipA, and C-terminal fragment of BipA with domains color-coded as in A. Residue number below the sequence indicates residue N-terminal to the boundary between domains. The beginning and end of disordered regions are indicated with the residue number of the last ordered residue on the top of the sequence. Disordered regions are displayed as an array of thin boxes. CTD, C-terminal domain. C, a representative section of the 2D Fo − mFc electron density map of apo full-length BipA contoured at 2.5 σ displayed in green mesh. D and E, unbiased Fo − Fo electron density map of the GDP-bound and ppGpp-bound BipA crystal structures, with GDP and ppGpp contoured at 4.0 σ in green mesh and the nucleotide-bound magnesium contoured at 6.0 σ in blue mesh.
Full-length protein of E. coli BipA crystallizes in the P21 space group with two copies in the asymmetric unit, but is monomeric in solution under physiologically relevant salt concentrations as demonstrated by gel filtration (data not shown). The structure was solved and refined to a resolution of 2.6 Å as described under “Experimental Procedures”. (For a representative section of the final electron density map, see Fig. 1C. For further data collection and crystallography statistics, see Table 1.)