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Switching aconitase B between catalytic and regulatory modes involves iron-dependent dimer formation
Yue Tang1, John R Guest, Peter J Artymiuk
1The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Molecular Microbiology
|May 11, 2005
Summary
Aconitase B (AcnB) protein homodimerization, regulated by iron, controls its gene regulatory function. The N-terminal domains of AcnB are key for both dimerization and mRNA binding, enabling iron-mediated switching between catalytic and regulatory roles.
Area of Science:
- Biochemistry
- Molecular Biology
- Gene Regulation
Background:
- Aconitase B (AcnB) in Escherichia coli functions as both a citric acid cycle enzyme and a post-transcriptional gene regulator.
- AcnB belongs to a unique aconitase superfamily branch, characterized by a HEAT-like domain (domain 5) involved in protein interactions.
Purpose of the Study:
- To investigate the molecular mechanisms underlying AcnB's homodimerization and its role in gene regulation.
- To identify the specific domains responsible for AcnB dimerization and iron-responsive interactions.
Main Methods:
- Gel filtration analysis to detect high-molecular-weight AcnB species.
- In vitro and in vivo protein interaction assays, including bacterial two-hybrid systems.
- Site-directed mutagenesis to create catalytically inactive AcnB variants (e.g., AcnB(C769S)).
- Analysis of N-terminal AcnB domains (AcnB5-4) for dimerization and mRNA binding capabilities.
Main Results:
- AcnB forms homodimers in cell-free extracts, and this dimerization is sensitive to iron availability.
- The N-terminal region (domains 4 and 5) of AcnB, not the iron-sulfur cluster domain (domain 3), mediates iron-responsive homodimerization.
- The isolated N-terminal polypeptide (AcnB5-4) is sufficient for dimerization and binds to the ftsH transcript, regulating flagella biosynthesis in Salmonella enterica.
Conclusions:
- Aconitase B utilizes an iron-mediated dimerization mechanism involving its N-terminal domains to switch between enzymatic and gene regulatory functions.
- The N-terminal region of AcnB is both necessary and sufficient for homodimer formation and target mRNA binding, providing a novel regulatory switch.