NMR Solution Structure and Extracellular Loop Dynamics of the Outer Membrane Protein OprG of Pseudomonas Aeruginosa Explain Transport of Small Amino Acids

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that is responsible for a growing number of nosocomial infections and the main cause of death in patients with cystic fibrosis. The structure of one of its outer membrane proteins (OprG) has been solved by x-ray crystallography and revealed a tall 8-stranded β-barrel that extends far into the extracellular space with a lumen lined with hydrophobic residues (Touw et al, PLoS ONE 5:15016[2011]). An interesting feature of that structure is a lateral proline-rich opening in the barrel wall near the membrane interface that has been suggested to function as a gate for small hydrophobic molecules that might subsequently diffuse across the bilayer of the outer membrane.In order to test this hypothesis we re-determined the structure of OprG in DHPC micelles by NMR. We show that the β-barrel has substantially shorter strands than in the crystal structure and longer extracellular loops without a defined lateral gate. To further investigate the role of the prolines in the proposed gate, we mutated them to alanines and determined the structure of the P92A mutant. Introduction of the P92A mutation resulted in an asymmetric elongation of the beta-barrel and changed the loop dynamics and the hydrogen-bonding pattern in the barrel-to-loop transition regions. As shown on a separate poster by PS, IK, and LKT, the physiological substrates of OprG are small amino acids and the P92A mutation aborts small amino acid transport.