An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets

Ofir Regev, Maayan Korman, Nir Hecht, Ziv Roth, Nadav Forer, Raz Zarivach, Eyal Gur

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Pupylation, the bacterial equivalent of ubiquitylation, involves the conjugation of a prokaryotic ubiquitin-like protein (Pup) to protein targets. In contrast to the ubiquitin system, where many ubiquitin ligases exist, a single bacterial ligase, PafA, catalyzes the conjugation of Pup to a wide array of protein targets. As mediators of target recognition by PafA have not been identified, it would appear that PafA alone determines pupylation target selection. Previous studies indicated that broad specificity and promiscuity are indeed inherent PafA characteristics that probably dictate which proteins are selected for degradation by the Pup-proteasome system. Nonetheless, despite the canonical role played by PafA in the Pup-proteasome system, the molecular mechanism that dictates target binding by PafA remains uncharacterized since the discovery of this enzyme about a decade ago. In this study, we report the identification of PafA residues involved in the binding of protein targets. Initially, docking analysis predicted the residues on PafA with high potential for target binding. Mutational and biochemical approaches subsequently confirmed these predictions and identified a series of additional residues located on an extended loop at the edge of the PafA active site. Mutating residues in this loop rendered PafA defective in the pupylation of a wide variety of protein targets but not in its catalytic mechanism, suggesting an important role for this extended loop in the binding of protein targets. As such, these findings pave the way toward an understanding of the molecular determinants that dictate the broad substrate specificity of PafA.

Original languageEnglish
Pages (from-to)4143-4153
Number of pages11
JournalJournal of Molecular Biology
Volume428
Issue number20
DOIs
StatePublished - 9 Oct 2016

Keywords

  • Dop
  • PafA
  • Pup
  • proteasome
  • proteolysis

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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