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Structural basis of substrate recognition by a polypeptide processing and secretion transporter.

Virapat Kieuvongngam1, Paul Dominic B Olinares2, Anthony Palillo1

  • 1Laboratory of Membrane Biophysics and Biology, The Rockefeller University, New York, United States.

Elife
|January 15, 2020
PubMed
Summary

Peptidase-containing ATP-binding cassette transporters (PCATs) process and export peptides. New cryo-EM structures reveal how PCAT1 recognizes its substrate CtA, coordinating cleavage and translocation for transport.

Keywords:
ABC transporterClostridium thermocellumcryoEMmembrane transportmolecular biophysicsprotein translocationstructural biology

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Peptidase-containing ATP-binding cassette transporters (PCATs) are ABC transporters that proteolytically process and export peptides.
  • PCATs possess peptidase, transmembrane, and nucleotide-binding domains crucial for their function.
  • Previous structures elucidated ATP's role in regulating PCAT protease activity and translocation access.

Purpose of the Study:

  • To determine the structural basis of substrate recognition by PCAT1.
  • To elucidate how the substrate CtA is recognized and processed by PCAT1.
  • To model the coordinated mechanism of substrate cleavage, ATP hydrolysis, and translocation.

Main Methods:

  • Determined the structure of the PCAT1-CtA complex using electron cryo-microscopy (cryo-EM).
  • Achieved a resolution of 3.4 Å for the complex structure.
  • Analyzed the binding mode of the substrate CtA to PCAT1.

Main Results:

  • The cryo-EM structure revealed two CtA molecules bound to PCAT1 via their N-terminal leader peptides.
  • Only one bound CtA molecule was observed in a position suitable for cleavage and translocation.
  • Provided insights into substrate recognition and initial processing steps.

Conclusions:

  • Proposed a model for the coordinated transport cycle involving substrate cleavage, ATP hydrolysis, and translocation.
  • The structure provides a mechanistic understanding of how PCATs recognize and initiate the processing of their peptide substrates.
  • This work advances our understanding of the intricate mechanisms governing ABC transporter function in protein export.