Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Structural insights into the bacterial outer-membrane transporter PqqU and how it scavenges PQQ from the environment (#309)

Fabian Munder 1 , Hari Venugopal 2 , Klaus Hantke 3 , Rhys Grinter 1
  1. Department of Microbiology, Monash University, Melbourne, Victoria, Australia
  2. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, Victoria, Australia
  3. Faculty of Science, University of Tuebingen, Tuebingen, Baden-Wuerttemberg, Germany

Gram-negative bacteria contain an inner and outer membrane (OM), the latter of which is a permeability barrier that prevents diffusion of molecules >600 Da. Bacteria utilize OM transporters to acquire large molecules as well as scarce micro-nutrients. Most OM transporters belong to the family of TonB-ExbBD-dependent proteins, which have a characteristic β-barrel fold.
Escherichia coli prefers glucose as a carbon source and imports glucose via the phosphotransferase system (PTS). Alternatively, the glucose dehydrogenase (Gcd) oxidizes glucose into gluconolactone which is imported into the cytosol via a separate pathway. The functionality of Gcd is dependent on pyrroloquinoline quinone (PQQ), a 330 Da redox cofactor. Interestingly, E. coli lacks the machinery for PQQ synthesis and instead scavenges PQQ with the membrane protein PqqU (PQQ uptake) from the environment.
In this study, we present the 2.3 Å cryo-electron microscopy structure of PqqU with bound PQQ. Upon substrate recognition, two surface-exposed loops undergo a significant conformational change, to enclose the PQQ binding site, which contains a single PQQ molecule coordinated by 10 amino acids. This isolates the PQQ binding site from the external environment in a gate-keeping mechanism to prevent the import of non-target molecules.
We used isothermal titration calorimetry to determine that PqqU binds PQQ with a high-affinity KD in the low nanomolar / high picomolar range, and a 1:1 molar ratio, which is consistent with our structural data.
To further characterize the binding pocket of PqqU, we generated expression plasmids with mutations of all PQQ interacting residues. In growth studies containing glucose as the only carbon source, we were able to rescue an E. coli Δpts ΔpqqU double-knockout strain by complementation with wildtype PqqU and we observed growth defects for complementation with mutated active site variations.
Based on the above findings, we propose a microbial community model for the function and evolutionof PqqU. Some bacteria favour PQQ-scavenging from within a microbial community over PQQ synthesis, presumably to save energy.