Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded β-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.

FIGURE 1.

Association of LPS with OprH in Pseudomonas outer membranes. The membrane lysates from PAO1 ΔoprH containing either the cloning vector pHERD30T (Empty Vector) or pHERD30T-oprH-FLAG (OprH-FLAG) were prepared and solubilized in 20 mm NaPO₄ at pH 7.4, 500 mm NaCl, 2% octyl-glucoside and 2.5 mm EDTA. A and B, Western blots of aliquots taken from the OM lysates. The aliquots were either left untreated (A) or treated (B) with proteinase K (10 μg/ml) prior to Western immunoblot analysis utilizing a polyclonal antibody specific for P. aeruginosa serogroup O5. C and D, after immunoprecipitation of OprH-FLAG, the resulting precipitate from each respective sample was immunoblotted with either anti-FLAG (C) or anti-LPS (D) monoclonal antibodies.

FIGURE 2.

Trypsin protection of OprH by LPS. A, SDS-PAGE gel showing trypsin digestion products under different conditions. 16 mm OprH in 20 mm Tris-HCl at pH 7.3 and 25 mm DHPC were incubated at 37 °C under the conditions shown for 5 h prior to running the gel. Samples without MgCl₂ also contained 5 mm EDTA. A schematic representation of the digestion products after treatment of OprH in DHPC micelles and DHPC:LPS mixed micelles with trypsin is shown on the bottom. Cleavage sites in the extracellular loops protected in both DHPC micelles and DHPC:LPS mixed micelles, as described under “Results,” are denoted (*) B, LPS concentration dependence of OprH protection from trypsin cleavage shown by SDS-PAGE. Samples contained 16 mm OprH in 20 mm Tris-HCl at pH 7.3, 25 mm DHPC, and LPS concentrations as indicated. C, the relative integral fractions of the 18- (■), 15- (●), or 8-kDa (▴) apparent molecular mass bands determined by SDS-PAGE gel densitometry are plotted against the LPS concentration. Error bars represent the standard deviations of five independent experiments.

FIGURE 3.

¹⁵N-¹H TROSY spectrum of ²H-,¹³C-,¹⁵N-labeled OprH in DHPC micelles collected at 800 MHz and 45 °C. The refolded protein sample was exchanged into 25 mm NaPO₄ at pH 6.1, 50 mm KCl, 0.05% NaN₃, and 5% D₂O before being concentrated to ∼1.0 mm for NMR experiments. Assignments determined as described under “Results” are shown. For some residues, all found in the micelle-solvent interfacial region of the protein, a second set of weaker peaks could be assigned. These residues are denoted with an apostrophe.

FIGURE 4.

Three-bond averaged secondary chemical shifts of OprH in DHPC micelles. The secondary chemical shifts, where the deviation (Δ) of each residue-specific Cα and Cβ chemical shift from random coil values was determined as (ΔC^α − ΔC^β) = 1/3 (ΔC^α_i−1 + ΔC^α_i + ΔC^α_i+1 − ΔC^β_i−1 − ΔC^β_i − ΔC^α_i+1), are plotted as a function of the amino acid sequence. Large negative values are indicative of β-sheet secondary structure, whereas large positive values are indicative of α-helical structure. Chemical shifts were corrected for both deuteration and TROSY effects prior to analysis (22). The secondary structure pattern observed in the solution structure is shown on the bottom.

FIGURE 5.

Topology schematic of OprH. Residues that were partially assigned are colored light gray, and residues that were completely unassigned are colored dark gray. For all other residues, complete nitrogen, hydrogen, Cα, Cβ, and CO assignments were obtained. Residues that face the lumen of the barrel are colored light blue. β-Strand residues are denoted as squares and were determined from the solution structure using the Kabsch and Sander secondary structure algorithm provided with MOLMOL software (46, 47). Loop and turn residues are denoted as circles. Inter-residue lines represent long- and medium-range NOEs observed in NOESY experiments. Hydrogen bond constraints that were identified through ²H/¹H exchange experiments are denoted as black inter-residue lines.

FIGURE 6.

Solution structure of OprH in DHPC micelles. A, NMR ensemble of the 20 lowest energy structures calculated. B, top-down view of the lowest energy conformer of OprH from the ensemble of 20 lowest energy structures. C, two side views of the lowest energy conformer with the side chains of aromatic residues (red) located in the ordered β-barrel region are shown. The β-barrel and loop/turn regions are colored blue and gray, respectively, in B and C.

FIGURE 7.

Backbone dynamics of OprH. A–C, the longitudinal relaxation times (A), transverse relaxation times (B), and {¹H}-¹⁵N heteronuclear NOEs (C) of OprH in DHPC micelles determined at 800 MHz and 45 °C are plotted as a function of the amino acid sequence. Blue bars in the T₁ and T₂ plots are the upper limits of the standard deviations. Blue bars in the NOE plot represent the upper limits of the standard errors. The secondary structure pattern observed in the solution structure is shown on the bottom.

FIGURE 8.

Effect of LPS on the amide backbone resonances of OprH in DHPC:LPS mixed micelles. A, chemical shift perturbations between ¹⁵N-¹H TROSY spectra of ²H-,¹³C-,¹⁵N-labeled OprH in DHPC micelles (150:1, DHPC:OprH molar ratio) and DHPC:LPS mixed micelles (150:10:1, DHPC:LPS:OprH molar ratio) in the presence of 5 mm EDTA were determined. These differences are shown as compound chemical shift changes (Δδ_comp = [Δδ²_HN + (Δδ_N/6.5) ²]½) (48) mapped color-coded onto the lowest energy structure of OprH in DHPC micelles. B, electrostatic surface potential plots of the lowest energy structure of OprH in DHPC micelles generated using the charge-smoothing algorithm in PyMOL (49).

FIGURE 9.

Trypsin accessibility and protection of the OprH extracellular loops in the presence of LPS. A, trypsin digestion products after treatment of 16 mm wild-type (WT) OprH and OprH loop deletion mutants (ΔL1–L4) in 20 mm Tris-HCl at pH 7.3, 25 mm DHPC, and 0.2 mm LPS with 0.7 mm trypsin in the presence of either 5 mm EDTA or 2 mm MgCl₂ and subsequent SDS-PAGE after sample boiling. B, schematic representation of the digestion products after treatment of OprH and OprH deletion mutants in DHPC:LPS mixed micelles with trypsin. The digestion products after treatment of OprH in DHPC micelles with trypsin are also shown for comparison. Potential trypsin cleavage sites in the extracellular loops are identified with arrows. Cleavage sites in the extracellular loops protected in both DHPC micelles and DHPC:LPS mixed micelles, as described under “Results,” are denoted (*).