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Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element.

Li YC, Chang CK, Chang CF, Cheng YH, Fang PJ, Yu T, Chen SC, Li YC, Hsiao CD, Huang TH - Nucleic Acids Res. (2014)

Bottom Line: The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion.Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner.Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.

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Dynamics of kpRstA DBD as probed by NMR. (A) Sequence variations of the 1H-, 15N-R1 (top), 15N-R2 (middle) and 15N NOE (bottom) of kpRstA DBD (filled circles) and kpRstA DBD/DNA-16a complex (open circles). (B) Sequence variations of the reduced spectral density functions J(0) (top), J(N) (middle) and J(0.87H) (bottom) deduced from the dynamic data shown in (A).
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Figure 7: Dynamics of kpRstA DBD as probed by NMR. (A) Sequence variations of the 1H-, 15N-R1 (top), 15N-R2 (middle) and 15N NOE (bottom) of kpRstA DBD (filled circles) and kpRstA DBD/DNA-16a complex (open circles). (B) Sequence variations of the reduced spectral density functions J(0) (top), J(N) (middle) and J(0.87H) (bottom) deduced from the dynamic data shown in (A).

Mentions: To gain further insights into the dynamic behavior of kpRstA DBD, we investigated the backbone dynamics of DNA-free kpRstA DBD and kpRstA DBD/DNA-16a complex by measuring 15N-R1, 15N-R2 and [1H–15N]-NOE at 800 MHz (Figure 7A) and calculating the reduced spectral density functions from these relaxation parameters (33,34,57,58). Reduced spectral density functions reflect the degree of motion in particular frequency regions. Thus, higher value of J(ω) indicates the presence of motion at the frequency ω MHz region. The results are shown in Figure 7B. J(0), J(N) and J(0.87H) represent reduced spectral density function at 0, 80 and 696 MHz, respectively. Overall, the DNA-free kpRstA DBD (filled circles) is rigid, except two loop regions. The loop connecting α2 and α3 (Lys187–Ser200) is characterized by large J(0.87H) indicative of the presence of high frequency fast motion. Another loop region showing flexibility is that connecting α3 and β4 (Ala216–Glu218). This region also showed elevated J(0.87H) suggesting the presence of fast motion. Moreover, this loop region also showed considerable slow motion, as indicated by large J(0). Interestingly, these two loop regions juxtapose helix α3. In the DNA-bound form (open circles), the DBD showed a similar dynamic behavior and the two flexible loops between Ala216–Glu218 and Lys187–Ser200 also exhibit higher flexibility as observed in the free form. However, reliable relaxation measurements cannot be obtained for many resonances between α2 and α3, indicating that this region becomes more flexible upon DNA-binding. One also observes higher and more scattered J(0) for the α3–β4 region, suggesting that the entire C-terminal region beyond α2 exhibit slow motion, possibly some conformational exchange process. The functional significance of the enhanced dynamics of the two regions is discussed in the following section. In agreement with the monomeric protein sizes, the overall rotational correlation times deduced from the relaxation data are 6.75 and 12.5 ns for the DNA-free and DNA-bound form, respectively.


Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element.

Li YC, Chang CK, Chang CF, Cheng YH, Fang PJ, Yu T, Chen SC, Li YC, Hsiao CD, Huang TH - Nucleic Acids Res. (2014)

Dynamics of kpRstA DBD as probed by NMR. (A) Sequence variations of the 1H-, 15N-R1 (top), 15N-R2 (middle) and 15N NOE (bottom) of kpRstA DBD (filled circles) and kpRstA DBD/DNA-16a complex (open circles). (B) Sequence variations of the reduced spectral density functions J(0) (top), J(N) (middle) and J(0.87H) (bottom) deduced from the dynamic data shown in (A).
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Figure 7: Dynamics of kpRstA DBD as probed by NMR. (A) Sequence variations of the 1H-, 15N-R1 (top), 15N-R2 (middle) and 15N NOE (bottom) of kpRstA DBD (filled circles) and kpRstA DBD/DNA-16a complex (open circles). (B) Sequence variations of the reduced spectral density functions J(0) (top), J(N) (middle) and J(0.87H) (bottom) deduced from the dynamic data shown in (A).
Mentions: To gain further insights into the dynamic behavior of kpRstA DBD, we investigated the backbone dynamics of DNA-free kpRstA DBD and kpRstA DBD/DNA-16a complex by measuring 15N-R1, 15N-R2 and [1H–15N]-NOE at 800 MHz (Figure 7A) and calculating the reduced spectral density functions from these relaxation parameters (33,34,57,58). Reduced spectral density functions reflect the degree of motion in particular frequency regions. Thus, higher value of J(ω) indicates the presence of motion at the frequency ω MHz region. The results are shown in Figure 7B. J(0), J(N) and J(0.87H) represent reduced spectral density function at 0, 80 and 696 MHz, respectively. Overall, the DNA-free kpRstA DBD (filled circles) is rigid, except two loop regions. The loop connecting α2 and α3 (Lys187–Ser200) is characterized by large J(0.87H) indicative of the presence of high frequency fast motion. Another loop region showing flexibility is that connecting α3 and β4 (Ala216–Glu218). This region also showed elevated J(0.87H) suggesting the presence of fast motion. Moreover, this loop region also showed considerable slow motion, as indicated by large J(0). Interestingly, these two loop regions juxtapose helix α3. In the DNA-bound form (open circles), the DBD showed a similar dynamic behavior and the two flexible loops between Ala216–Glu218 and Lys187–Ser200 also exhibit higher flexibility as observed in the free form. However, reliable relaxation measurements cannot be obtained for many resonances between α2 and α3, indicating that this region becomes more flexible upon DNA-binding. One also observes higher and more scattered J(0) for the α3–β4 region, suggesting that the entire C-terminal region beyond α2 exhibit slow motion, possibly some conformational exchange process. The functional significance of the enhanced dynamics of the two regions is discussed in the following section. In agreement with the monomeric protein sizes, the overall rotational correlation times deduced from the relaxation data are 6.75 and 12.5 ns for the DNA-free and DNA-bound form, respectively.

Bottom Line: The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion.Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner.Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.

Show MeSH
Related in: MedlinePlus