An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets
| Autor: | Ofir Regev, Nadav Forer, Ziv Roth, Eyal Gur, Nir Hecht, Raz Zarivach, Maayan Korman |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Proteolysis DNA Mutational Analysis 010402 general chemistry 01 natural sciences Ligases 03 medical and health sciences Ubiquitin Structural Biology medicine Ubiquitins Molecular Biology chemistry.chemical_classification DNA ligase biology medicine.diagnostic_test Active site 0104 chemical sciences Cell biology Corynebacterium glutamicum Molecular Docking Simulation 030104 developmental biology Enzyme Proteasome Biochemistry Pupylation chemistry Docking (molecular) biology.protein Protein Binding |
| Zdroj: | Journal of Molecular Biology. 428:4143-4153 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2016.07.021 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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