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Rapid Generation of Amyloid from Native Proteins In vitro
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Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Xuan Wang1, Yizhou Zhou, Juan-Jie Ren

  • 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.

Proceedings of the National Academy of Sciences of the United States of America
|December 8, 2009
PubMed

Insights

Bacterial amyloid curli fibers are regulated by specific "gatekeeper" residues in CsgA. Removing these residues causes uncontrolled polymerization, leading to cytotoxicity in Escherichia coli.

Area of Science:

  • Microbiology
  • Biochemistry
  • Structural Biology

Background:

  • Amyloid fibers are protein structures linked to neurodegenerative diseases.
  • Escherichia coli produce functional amyloid fibers called curli, essential for bacterial adhesion and biofilm formation.
  • Curli assembly involves the CsgA protein, whose polymerization is nucleated by CsgB.

Purpose of the Study:

  • To investigate the specificity of bacterial amyloid nucleation.
  • To identify residues controlling CsgA polymerization and responsiveness to nucleation.
  • To understand the role of these residues in modulating curli fiber formation and cellular toxicity.

Main Methods:

  • Analysis of CsgA protein structure, focusing on repeat regions (R1-R5).
  • Site-directed mutagenesis to remove identified 'gatekeeper' residues (aspartic acid and glycine).
  • In vitro polymerization assays and in vivo studies in Escherichia coli to assess CsgA polymerization kinetics and localization.

Main Results:

  • Specific aspartic acid and glycine residues in CsgA repeats R2-R4 act as gatekeepers, inhibiting intrinsic aggregation.
  • Mutant CsgA lacking gatekeeper residues exhibited significantly faster in vitro polymerization.
  • In vivo, mutant CsgA polymerized without CsgB nucleation, forming mislocalized fibers and causing cytotoxicity.

Conclusions:

  • Gatekeeper residues in CsgA are crucial for regulating curli fiber assembly and preventing uncontrolled polymerization.
  • Dysregulation of CsgA polymerization through removal of gatekeeper residues leads to detrimental effects on bacterial cells.
  • This study highlights the importance of precise control over bacterial amyloid formation for cellular viability.

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