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Cryo-EM structure of the bacterial intramembrane metalloprotease RseP in the substrate-bound state

Kikuko Asahi1, Mika Hirose2, Rie Aruga1

  • 1Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

Science Advances
|February 26, 2025
PubMed

Insights

Site-2 proteases (S2Ps) are crucial for cellular homeostasis and linked to bacterial multidrug resistance. This study reveals how these metalloproteases accommodate substrates by threading them through a membrane cavity, aiding antimicrobial drug development.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Microbiology

Background:

  • Site-2 proteases (S2Ps) are conserved intramembrane metalloproteases vital for cellular homeostasis.
  • S2Ps are implicated in bacterial chronic infections and the development of multidrug resistance.
  • Understanding S2P substrate recognition is key to developing novel antimicrobial agents.

Purpose of the Study:

  • To elucidate the mechanism by which S2Ps accommodate full-length membrane-spanning substrates.
  • To provide a structural model for substrate binding and discrimination by S2Ps.
  • To inform the design of selective antimicrobial agents targeting bacterial S2Ps.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) analysis of Aquifex aeolicus RseP (AaRseP).
  • In vivo photocrosslinking experiments.
  • Structural modeling of substrate accommodation.

Main Results:

  • Cryo-EM revealed a substrate-like membrane protein fragment occupying the AaRseP active site within a transmembrane cavity.
  • This cavity is inaccessible via lateral diffusion, suggesting a threading mechanism.
  • In vivo photocrosslinking confirmed this substrate accommodation mode on the cell membrane.
  • Substrate threading through a conserved membrane-associated region stabilizes the complex and aids discrimination.

Conclusions:

  • Substrate accommodation in S2Ps involves threading through a conserved membrane-associated region.
  • This mechanism stabilizes the substrate-protease complex and is critical for substrate discrimination.
  • The findings provide insights into S2P function and potential targets for antimicrobial drug development.

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