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FepA with globular domain deletions lacks activity
Hema L Vakharia1, Kathleen Postle
1School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234, USA.
Journal of Bacteriology
|September 10, 2002
Summary
TonB-gated transporters utilize a globular domain for ligand transport. Deleting this domain in FepA unexpectedly restored function in specific strains, suggesting protein complementation may restore TonB-dependent activity.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- TonB-gated transporters feature a beta-barrel with an amino-terminal globular domain crucial for occluding the barrel's interior.
- Mutations within this globular domain typically inhibit ligand transport across the outer membrane.
- Previous studies suggested FepA with globular domain deletions retained partial function, contradicting established mechanisms.
Purpose of the Study:
- To investigate the role of the FepA globular domain in TonB-dependent transport.
- To understand the interaction between TonB and the beta-barrel transporter structure.
- To reconcile conflicting findings regarding FepA globular domain deletions.
Main Methods:
- Constructing and expressing FepA proteins with various deletions in the globular domain (amino acids 1-152, 7-152, 20-152, 17-150) in a deltafepA strain.
- Assessing the sensitivity of these deletion mutants to colicins B and D.
- Evaluating the binding of colicins and in vivo cross-linking to TonB.
- Testing mutant activity in different bacterial strains, including RWB18-60.
Main Results:
- FepA deletion mutants constructed in this study did not retain sensitivity to colicin B and showed only marginal sensitivity to colicin D.
- These mutants failed to bind colicin B and did not detectably cross-link to TonB in vivo.
- Activity of the FepA globular domain deletion proteins was restored in the RWB18-60 strain.
- This restoration of function in specific strains suggests a potential role for interprotein complementation.
Conclusions:
- The FepA globular domain is essential for proper TonB-dependent transport and TonB interaction.
- Discrepancies with previous studies may be due to strain-specific factors.
- Interprotein complementation between nonfunctional proteins might restore TonB-dependent activity, offering a new perspective on transporter function.