Structural and functional insights into Klebsiella pneumoniae protein adenylyltransferase revealed by biophysical and computational analyses: a proof-of-concept study

Abstract

Hospital-acquired infections caused by multidrug-resistant Klebsiella pneumoniae remain a pressing global health challenge, highlighting the need for novel therapeutic targets. Protein adenylyltransferases (PrATs) catalyse AMPylation, a post-translational modification that may regulate redox-associated proteins, although the structural and mechanistic properties of K. pneumoniae PrAT ( Kp PrAT) remain largely unexplored. Here, we provide a proof-of-concept study integrating biochemical, biophysical, and computational approaches, including homology modelling, 1000 ns molecular dynamics simulations, and free energy landscapes analyses, to characterise Kp PrAT. Using bovine myelin basic protein as a model protein substrate, extrinsic fluorescence spectroscopy and isothermal titration calorimetry confirmed substrate binding and Mg 2+ -dependent stabilisation of the ATP-binding site. LC-MS/MS validated AMPylation at tyrosine 12 of the protein substrate, while molecular dynamics simulations and free energy landscape analyses revealed ligand-dependent conformational dynamics, including transitions between compact and flexible states critical for catalysis. These combined analyses allowed the proposal of a catalytic cycle involving ATP binding, AMP transfer, pyrophosphate release, and enzyme regeneration. Collectively, our findings provide the first mechanistic insights into Kp PrAT, highlighting its dynamic regulation and establishing AMPylation pathways as potential therapeutic targets against multidrug-resistant K. pneumoniae .