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Substrates bind to residues lining the ring of asymmetrically engaged bacterial proteasome activator Bpa

Tatjana von Rosen1, Rafal Zdanowicz1,2, Yasser El Hadeg1

  • 1Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.

Nature Communications
|March 29, 2025
PubMed

Insights

Researchers visualized the Mycobacterium tuberculosis proteasome activator Bpa interacting with its substrate HspR using cryo-EM. This reveals key interactions for protein degradation, crucial for bacterial persistence.

Area of Science:

  • Microbiology
  • Structural Biology
  • Biochemistry

Background:

  • Mycobacteria possess a unique proteasome acquired via horizontal gene transfer, essential for the survival of Mycobacterium tuberculosis in host macrophages.
  • The proteasome core particle (20S CP) functions with activator complexes to degrade proteins, including the heat shock repressor HspR, via the bacterial proteasome activator Bpa.
  • Bpa facilitates ATP-independent degradation of HspR, a critical process for bacterial persistence.

Purpose of the Study:

  • To determine the high-resolution 3D structure of the complex formed between the bacterial proteasome activator Bpa and its substrate HspR.
  • To elucidate the molecular interactions governing Bpa-mediated substrate recognition and proteasomal degradation.
  • To understand the structural basis of Bpa's interaction with the 20S proteasome core particle.

Main Methods:

  • Cryogenic electron microscopy (cryo-EM) was employed to obtain a 3D reconstruction of the Bpa-HspR complex at 4.1 Å resolution.
  • Structure-guided site-directed mutagenesis was performed to investigate the functional significance of identified interaction residues.
  • In vitro biochemical assays were utilized to confirm the role of specific residues in substrate recruitment and degradation.

Main Results:

  • The cryo-EM structure revealed specific regions of Bpa interacting with HspR, providing insights into substrate binding.
  • Mutagenesis and biochemical assays confirmed the importance of identified residues for Bpa's function in recruiting HspR and promoting its degradation.
  • The dodecameric Bpa ring was observed to associate asymmetrically with the heptameric α-rings of the 20S CP, forming a 'hinged lid' conformation while engaging all docking sites.

Conclusions:

  • The study provides the first structural insights into the Bpa-HspR interaction, detailing how the proteasome activator recruits its substrate.
  • The findings highlight the critical role of specific amino acid residues in mediating substrate recognition and degradation by the mycobacterial proteasome.
  • The asymmetric association of Bpa with the 20S CP suggests a unique mechanism for regulating proteasomal activity in Mycobacterium tuberculosis.

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