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Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway

Dennis Özcelik1, Jonas Barandun, Nikolaus Schmitz

  • 1ETH Zurich, Institute of Molecular Biology & Biophysics, CH-8093, Switzerland.

Nature Communications
|August 23, 2012
PubMed

Insights

Pupylation, a protein modification in bacteria, is structurally and functionally similar to ubiquitination. This study reveals the crystal structures of key pupylation enzymes, PafA and Dop, detailing their unique folds and active sites.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Pupylation is a posttranslational modification in actinobacteria, analogous to ubiquitination.
  • Understanding pupylation enzymes is crucial for deciphering bacterial protein regulation.

Purpose of the Study:

  • To determine the crystal structures of PafA (Pup ligase) and Dop (depupylase/deamidase).
  • To elucidate the structural basis for pupylation and de-pupylation reactions.

Main Methods:

  • X-ray crystallography to obtain high-resolution structures of PafA and Dop.
  • Nuclear magnetic resonance (NMR) and biochemical assays to identify protein interaction interfaces.
  • Site-directed mutagenesis to investigate key catalytic residues.

Main Results:

  • The crystal structures of PafA and Dop reveal a conserved fold with unique domains and an active site pocket.
  • A conserved groove near the active site is proposed to be involved in Pup binding.
  • NMR and biochemical data pinpoint the interacting regions of Pup with PafA and Dop.
  • Mutational studies identified critical residues essential for the catalytic activity of both enzymes.

Conclusions:

  • The structural and biochemical data provide a detailed molecular understanding of the pupylation pathway.
  • These findings offer insights into the functional analogy between pupylation and ubiquitination.
  • The identified structures and crucial residues pave the way for future functional and therapeutic studies.

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