Mapping the structural heterogeneity of Pup ligase PafA using H/D exchange mass spectrometry
Abstract
The Pup-proteasome system (PPS) is a unique bacterial proteolytic pathway found in some bacterial species, including in Mycobacterium tuberculosis, that plays a vital role in maintaining proteome integrity and survival during infection. Pupylation is the process of tagging substrates with Pup for degradation and is catalyzed by PafA, the sole Pup ligase in bacteria. However, how PafA interacts with diverse targets and its oligomeric state remains poorly understood. Although X-ray crystal structures have characterized PafA as a domain-swapped dimer, it is widely regarded as functionally active in its monomeric form. It remains to be established whether PafA dimerizes in solution, and how dimerization influences its function. In this study, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside complementary biophysical techniques to explore the oligomeric states and conformational dynamics of PafA. We show that recombinantly-produced PafA exists in a monomeric and a domain-swapped dimeric state in solution. Although nucleotide binding stabilizes PafAdimer, it primarily adopts a catalytically inactive conformation. Our HDX-MS highlighted regions throughout the N- and C-terminal domains that facilitate the PafA dimerization process. HDX-MS also revealed nucleotide binding induces global conformational changes on PafAmonomer, underscoring the structural plasticity of this promiscuous enzyme. Our findings enhance our understanding of the structural and…
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