Probing the structural heterogeneity of Pup ligase PafA using H/D exchange mass spectrometry

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 remain 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. While PafA _dimer is stabilized by nucleotide, it predominately resides in 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 the PafA _monomer , underscoring the structural plasticity of this promiscuous enzyme. Our findings provide insight into the structure-function-dynamics relationship of PafA, shedding light on how its oligomeric states and conformational flexibility might influence its role in the PPS.