Related Experiment Videos
Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB
H H Kimsey1, M D Dagarag, C A Kumamoto
1Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site.
Insights
Escherichia coli SecB protein mutations reveal distinct roles in preMBP binding and export. Specific substitutions affect complex stability, while others impact export rate, suggesting regulatory functions in protein translocation.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Cellular Transport
Background:
- SecB protein in Escherichia coli facilitates the cytoplasmic export of precursor maltose-binding protein (preMBP).
- Understanding the SecB-preMBP interaction is crucial for elucidating protein secretion pathways.
Purpose of the Study:
- To investigate the molecular determinants of the SecB-preMBP interaction using site-directed mutagenesis.
- To differentiate the roles of specific SecB regions in preMBP binding affinity and export efficiency.
Main Methods:
- Site-directed mutagenesis of the Escherichia coli SecB protein.
- Analysis of 30 aminoacyl substitution mutants.
- Assessment of SecB.preMBP complex formation and MBP export rates in vivo.
Main Results:
- Two classes of secB mutants were identified based on their functional defects.
- Mutations in hydrophobic residues (Phe-74, Cys-76, Val-78, Gln-80) weakened SecB.preMBP complex stability but had mild effects on export rate.
- Mutations in acidic residues (Asp-20, Glu-24, Leu-75, Glu-77) severely impaired MBP export rate without disrupting complex formation.
Conclusions:
- A hydrophobic region with beta-sheet structure likely constitutes a primary binding site for preMBP.
- Acidic residues in SecB may regulate the preprotein binding site, balancing high-affinity binding with efficient dissociation for membrane translocation.