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Related Experiment Videos

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB.

G S Anand1, P N Goudreau, J K Lewis

  • 1Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.

Protein Science : a Publication of the Protein Society
|June 13, 2000
PubMed
Summary

Phosphorylation of the CheB response regulator activates its methylesterase activity by inducing conformational changes. These changes reposition regulatory domains, enhancing substrate access and enzyme function.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • The response regulator CheB's methylesterase activity is regulated by phosphorylation.
  • The N-terminal regulatory domain has dual roles: inhibitory when unphosphorylated and stimulatory when phosphorylated.
  • Structural studies suggest inhibition results from blocked substrate access to the active site.

Purpose of the Study:

  • To investigate phosphorylation-dependent conformational changes in CheB.
  • To provide evidence supporting a model of CheB activation involving domain repositioning.

Main Methods:

  • Comparative analysis of proteolytic cleavage rates by trypsin.
  • Examining phosphorylated versus unphosphorylated states of CheB.
  • Identifying specific cleavage sites (113, 134, 148) within CheB.

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Main Results:

  • Observed differences in trypsin cleavage rates between phosphorylated and unphosphorylated CheB.
  • Identified three distinct proteolytic cleavage sites.
  • One site (113) is in the regulatory domain, and two sites (134, 148) are in the interdomain linker.

Conclusions:

  • Results support a mechanism where N-terminal phosphorylation triggers conformational changes.
  • These changes propagate within the regulatory domain, leading to domain repositioning.
  • Repositioning of N- and C-terminal domains is crucial for stimulating CheB methylesterase activity.