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Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli.

Fan H, Hahm J, Diggs S, Perry JJ, Blaha G - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of California, Riverside, California 92521.

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Related in: MedlinePlus

Structure of C-terminal fragment of BipA consisting of domain III, V, and C-terminal domain.A, overall view of the C-terminal fragment with protein rendered as a schematic and ligated magnesium ions as green spheres. CTD, C-terminal domain. B, surface charge distribution of the C-terminal domain within the C-terminal fragment with positive charges displayed in blue and negative charges displayed in red. The C-terminal helix essential for ribosome binding is outlined with a dashed line. C, 2D Fo − mFc electron density map of the two proximal magnesium ions (green spheres with coordinated water molecules as red spheres) bound to the C-terminal domain of C-terminal fragment (blue) contoured at 1.0 σ in green mesh. The pink sphere indicates the position of cobalt ion bound to the C-terminal domain of the full-length BipA (light gray). D, superposition of the C-terminal fragment by itself and that derived from the full-length BipA structure. The domains of the C-terminal fragment structure are displayed in the same colors as in Fig. 1, except that all domains of the derived C-terminal fragment are displayed in light gray.
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Figure 2: Structure of C-terminal fragment of BipA consisting of domain III, V, and C-terminal domain.A, overall view of the C-terminal fragment with protein rendered as a schematic and ligated magnesium ions as green spheres. CTD, C-terminal domain. B, surface charge distribution of the C-terminal domain within the C-terminal fragment with positive charges displayed in blue and negative charges displayed in red. The C-terminal helix essential for ribosome binding is outlined with a dashed line. C, 2D Fo − mFc electron density map of the two proximal magnesium ions (green spheres with coordinated water molecules as red spheres) bound to the C-terminal domain of C-terminal fragment (blue) contoured at 1.0 σ in green mesh. The pink sphere indicates the position of cobalt ion bound to the C-terminal domain of the full-length BipA (light gray). D, superposition of the C-terminal fragment by itself and that derived from the full-length BipA structure. The domains of the C-terminal fragment structure are displayed in the same colors as in Fig. 1, except that all domains of the derived C-terminal fragment are displayed in light gray.

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%.


Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli.

Fan H, Hahm J, Diggs S, Perry JJ, Blaha G - J. Biol. Chem. (2015)

Structure of C-terminal fragment of BipA consisting of domain III, V, and C-terminal domain.A, overall view of the C-terminal fragment with protein rendered as a schematic and ligated magnesium ions as green spheres. CTD, C-terminal domain. B, surface charge distribution of the C-terminal domain within the C-terminal fragment with positive charges displayed in blue and negative charges displayed in red. The C-terminal helix essential for ribosome binding is outlined with a dashed line. C, 2D Fo − mFc electron density map of the two proximal magnesium ions (green spheres with coordinated water molecules as red spheres) bound to the C-terminal domain of C-terminal fragment (blue) contoured at 1.0 σ in green mesh. The pink sphere indicates the position of cobalt ion bound to the C-terminal domain of the full-length BipA (light gray). D, superposition of the C-terminal fragment by itself and that derived from the full-length BipA structure. The domains of the C-terminal fragment structure are displayed in the same colors as in Fig. 1, except that all domains of the derived C-terminal fragment are displayed in light gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543647&req=5

Figure 2: Structure of C-terminal fragment of BipA consisting of domain III, V, and C-terminal domain.A, overall view of the C-terminal fragment with protein rendered as a schematic and ligated magnesium ions as green spheres. CTD, C-terminal domain. B, surface charge distribution of the C-terminal domain within the C-terminal fragment with positive charges displayed in blue and negative charges displayed in red. The C-terminal helix essential for ribosome binding is outlined with a dashed line. C, 2D Fo − mFc electron density map of the two proximal magnesium ions (green spheres with coordinated water molecules as red spheres) bound to the C-terminal domain of C-terminal fragment (blue) contoured at 1.0 σ in green mesh. The pink sphere indicates the position of cobalt ion bound to the C-terminal domain of the full-length BipA (light gray). D, superposition of the C-terminal fragment by itself and that derived from the full-length BipA structure. The domains of the C-terminal fragment structure are displayed in the same colors as in Fig. 1, except that all domains of the derived C-terminal fragment are displayed in light gray.
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%.

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.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of California, Riverside, California 92521.

Show MeSH
Related in: MedlinePlus