Abstract
Truncated pilin monomers from Pseudomonas aeruginosa strain K122-4 (ΔK122) have been shown to enter a monomer-dimer equilibrium in solution prior to oligomerization into protein nanotubes. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved hydrogen-deuterium exchange mass spectrometry. Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state.
Highlights
Opportunistic infections by Pseudomonas aeruginosa are prevalent in patients with compromised immune systems including those recovering from burn wounds1–3 and organ transplants,4,5 as well as in individuals suffering from cystic fibrosis,6,7 acute leukemia,8 and HIV.9These persistent infections are initiated by interaction of a type IV pilus (T4P) with receptors on the mucosal cells of susceptible hosts.10–15 T4P are responsible for a variety of bacterial processes including surface motility,14,16–19 microcolony and biofilm formation,14,20–23 cell-host adhesion,15 cell signalling,24 and DNA uptake.25,26 T4P are important structures found across a wide range of gram-negative and gram-positive bacteria, and disruption of the pilus leads to decreased bacterial virulence in many gram-negative pathogens.2,13,14,27,28The type IV pilus is a filamentous protein polymer of single monomeric unit, the type 4 pilin
We examine the structural changes occurring between the monomeric and dimeric states of DK122 using time-resolved hydrogen-deuterium exchange mass spectrometry
Understanding the structural changes that occur when the protein enters its dimeric state is of great importance as it is the earliest intermediate leading up to protein nanotube formation
Summary
Opportunistic infections by Pseudomonas aeruginosa are prevalent in patients with compromised immune systems including those recovering from burn wounds and organ transplants, as well as in individuals suffering from cystic fibrosis, acute leukemia, and HIV.9 These persistent infections are initiated by interaction of a type IV pilus (T4P) with receptors on the mucosal cells of susceptible hosts.. A truncated form of the pilin from P. aeruginosa strain K122-4 (DK122) lacking the conserved a1-N region of the helix has been shown to form T4Plike structures, so called protein nanotubes (PNTs), both in solution and at surfaces.48,53–55 As this truncated form of the pilin lacks the predicted main driving force for pilus oligomerization, namely the conserved a1-N region, other protein-protein interactions are required to stabilize the. Understanding the structural changes that occur in this equilibrium state will shed light on the earliest intermediate leading up to fibril formation and PNT oligomerization
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