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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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ITC and SAXS analysis of nucleotide binding to BipA.A and B, isothermal titration calorimetry curves of BipA titrated with GDP (A) and ppGpp (B) after correction of the dilution effect of nucleotides. C, enthalpy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of temperature (°C). D, entropy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of the logarithm of temperature in Kelvin. The heat capacity change (ΔCp) value from the enthalpy (C) is similar to that obtained from entropy (D). E, the unique scattering profiles of the apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are shown with intensity (I), plotted against the photon momentum transfer (q). F, the P(r) distribution functions of apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are depicted.
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Figure 4: ITC and SAXS analysis of nucleotide binding to BipA.A and B, isothermal titration calorimetry curves of BipA titrated with GDP (A) and ppGpp (B) after correction of the dilution effect of nucleotides. C, enthalpy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of temperature (°C). D, entropy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of the logarithm of temperature in Kelvin. The heat capacity change (ΔCp) value from the enthalpy (C) is similar to that obtained from entropy (D). E, the unique scattering profiles of the apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are shown with intensity (I), plotted against the photon momentum transfer (q). F, the P(r) distribution functions of apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are depicted.

Mentions: The switch of the binding preference of BipA from ribosomes to the small ribosomal subunits during starvation suggests a possible conformational change of BipA upon binding of ppGpp (11). We used ITC to estimate binding affinities, enthalpies, and entropies for the binding of ppGpp and of GDP to E. coli BipA at temperature intervals of 5–30 and 10–30 °C, respectively (Table 2). A typical titration curve for GDP and for ppGpp is shown in Fig. 4, A and B, respectively. The binding affinity of BipA for GDP is only slightly stronger than that for ppGpp. Plotting the measured binding enthalpy values against temperature yields the heat capacity change (ΔCp) due to nucleotide binding from the slope. The ΔCp estimates for GDP and ppGpp binding to BipA are −39.3 and −94.3 cal mol−1 K−1, respectively (Fig. 4C). Similar values of ΔCp for GDP and ppGpp binding were obtained using the temperature dependence of the binding entropy (Fig. 4D). On the assumption of additive contributions of partial heat capacities (31), a comparison of ΔCp upon binding of GDP and of ppGpp allows us to ascertain the contribution of the 3′-pyrophosphate group to ΔCp for ppGpp binding with −55.0 cal mol−1 K−1.


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)

ITC and SAXS analysis of nucleotide binding to BipA.A and B, isothermal titration calorimetry curves of BipA titrated with GDP (A) and ppGpp (B) after correction of the dilution effect of nucleotides. C, enthalpy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of temperature (°C). D, entropy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of the logarithm of temperature in Kelvin. The heat capacity change (ΔCp) value from the enthalpy (C) is similar to that obtained from entropy (D). E, the unique scattering profiles of the apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are shown with intensity (I), plotted against the photon momentum transfer (q). F, the P(r) distribution functions of apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: ITC and SAXS analysis of nucleotide binding to BipA.A and B, isothermal titration calorimetry curves of BipA titrated with GDP (A) and ppGpp (B) after correction of the dilution effect of nucleotides. C, enthalpy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of temperature (°C). D, entropy change for the GDP (open circles) and ppGpp binding (filled circles) to BipA as a function of the logarithm of temperature in Kelvin. The heat capacity change (ΔCp) value from the enthalpy (C) is similar to that obtained from entropy (D). E, the unique scattering profiles of the apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are shown with intensity (I), plotted against the photon momentum transfer (q). F, the P(r) distribution functions of apo BipA, in yellow, BipA with bound GDP, in green, and BipA with bound ppGpp, in blue, are depicted.
Mentions: The switch of the binding preference of BipA from ribosomes to the small ribosomal subunits during starvation suggests a possible conformational change of BipA upon binding of ppGpp (11). We used ITC to estimate binding affinities, enthalpies, and entropies for the binding of ppGpp and of GDP to E. coli BipA at temperature intervals of 5–30 and 10–30 °C, respectively (Table 2). A typical titration curve for GDP and for ppGpp is shown in Fig. 4, A and B, respectively. The binding affinity of BipA for GDP is only slightly stronger than that for ppGpp. Plotting the measured binding enthalpy values against temperature yields the heat capacity change (ΔCp) due to nucleotide binding from the slope. The ΔCp estimates for GDP and ppGpp binding to BipA are −39.3 and −94.3 cal mol−1 K−1, respectively (Fig. 4C). Similar values of ΔCp for GDP and ppGpp binding were obtained using the temperature dependence of the binding entropy (Fig. 4D). On the assumption of additive contributions of partial heat capacities (31), a comparison of ΔCp upon binding of GDP and of ppGpp allows us to ascertain the contribution of the 3′-pyrophosphate group to ΔCp for ppGpp binding with −55.0 cal mol−1 K−1.

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