Structural Basis for the Interaction of Lipopolysaccharide with Outer Membrane Protein H (OprH) from Pseudomonas aeruginosa

Abstract

Background: Pseudomonas aeruginosa outer membrane protein OprH has been hypothesized to confer antibiotic resistance by interaction with LPS. Results: The structure of OprH was solved and LPS interaction was demonstrated by solution NMR supported by pulldown and biochemical assays. Conclusion: OprH forms a ␤-barrel in membrane and interacts with LPS in vivo and in vitro. Significance: Structure and lipid interactions may help understand antibiotic resistance.

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

“…Previously published protocols for expression, purification, and folding were followed. 3 OprH was extracted in 50 mM sodium phosphate, pH 8, 300 mM NaCl, 20 mM imidazole, and purified over a Ni 21 IMAC column with 15 CV of wash buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl, 20 mM imidazole, and 8M urea) followed by 5 CV of elution buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 mM imidazole, and 8M urea). After purification, OprH was concentrated to 400 lM and diluted 10-fold into OprH refolding buffer (20 mM Tris-HCl, pH 8.5, 5 mM EDTA, 600 mM L-arginine with 3% 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, Anatrace)) and incubated at 310 C for 72 h. The diluted sample was concentrated and dialyzed against 2.5 L of 20 mM Tris-HCl, pH 8.5, 5 mM EDTA, and 50 mM KCl.…”

“…With these advances, six solution NMR b-barrel membrane protein structures (varying in the number of b-strands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and molecular weight (16-31 kDa) have been determined. [1][2][3][4][5][6] In this study, the difficulties of assigning Opa 60 , a b-barrel membrane protein from Neisseria gonorrhoeae, 22 are outlined and strategies for circumventing these challenges are demonstrated. Opa 60 is a putative eight-stranded b-barrel with four extracellular loops, which comprise approximately 63% of the protein.…”

“…To date, there are six NMR solution structures of b-barrel membrane proteins [1][2][3][4][5][6] (compared to the 89 unique b-barrel structures deposited in the Protein Data Bank), all except for OprH have corresponding X-ray crystal structures. [7][8][9][10][11][12] The first b-barrel membrane protein structure determined with solution NMR was in 2001 4 and the latest structure was determined in 2011 3 yielding an average of 0.6 structures a year. The dearth of solution NMR membrane protein structures is likely due to the difficulty of obtaining quality NMR spectra, which interferes with assigning the resonances and obtaining quality structural restraints.…”

“…[1][2][3][4][5][6] However, the number of high-resolution polytopic membrane protein structures is limited. To date, there are six NMR solution structures of b-barrel membrane proteins [1][2][3][4][5][6] (compared to the 89 unique b-barrel structures deposited in the Protein Data Bank), all except for OprH have corresponding X-ray crystal structures.…”

“…[1][2][3][4][5][6] However, the number of high-resolution polytopic membrane protein structures is limited. To date, there are six NMR solution structures of b-barrel membrane proteins [1][2][3][4][5][6] (compared to the 89 unique b-barrel structures deposited in the Protein Data Bank), all except for OprH have corresponding X-ray crystal structures. [7][8][9][10][11][12] The first b-barrel membrane protein structure determined with solution NMR was in 2001 4 and the latest structure was determined in 2011 3 yielding an average of 0.6 structures a year.…”