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