Solution structure and dynamics of the outer membrane enzyme PagP by NMR

Abstract

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-beta-d-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel beta-barrel preceded by an amphipathic alpha helix. The beta-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual beta-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.

Figure 1

(A) Alignment of PagP from five different pathogenic Gram-negative bacterial genera. Only representative sequences from each genus are shown when multiple species exist. The alignment was constructed after removal of signal peptides, either known (10) or predicted by signalp (44). The three residues shown to be essential for catalysis by site-directed mutagenesis are highlighted in red with yellow shading. Absolutely conserved residues are shaded in blue, and highly conserved residues in gray. The Yersinia pestis homologue appears to contain a premature stop codon at position 174. (B) PagP-catalyzed palmitoylation of lipid A. PagP can transfer a palmitate chain from the sn-1 position of a phospholipid, such as phosphatidylethanolamine (PtdEtn), to the free hydroxyl-group of the N-linked R-3-hydroxymyristate chain on the proximal glucosamine unit of lipid A or its precursors (10). The simplest lipid A acceptor for PagP in the outer membrane contains two units of 3-deoxy-d-manno-octulosonic acid (Kdo) and is known as Kdo₂-lipid A (10).

Figure 2

Expression and activity of PagP-His₆ and its site-directed mutant derivatives. (A) Membrane proteins were analyzed by SDS/PAGE and stained with Coomassie blue dye (10). The band corresponding to fully folded PagP is indicated. Note that unfolded PagP migrates more rapidly and did not accumulate for any of the mutants. (B) Palmitoyl transferase reactions were performed with induced membranes from the host strain BL21(DE3)pLysE at 100 ng/ml using ³²P-lipid IV_A as the acyl-acceptor, and the production of the palmitoylated metabolite lipid IV_B was detected by TLC (10). The compounds on the plate were visualized by overnight exposure to a PhosphorImager (Molecular Dynamics) screen. pET21a⁺-transformed cells were used as a negative control, whereas pETCrcAH-transformed cells expressing wild-type PagP served as a positive control.

Figure 3

¹H-¹⁵N TROSY-HSQC spectrum of 0.8 mM PagP-DPC, 45°C, at 800 MHz.

Figure 4

(A) Superposition of the lowest energy PagP-DPC structure (blue) and the lowest energy PagP-β-OG structure (red). The side chains of the residues implicated in catalysis, His-33, Asp-76, and Ser-77, are shown in green. (B) Stereoview of the 20 lowest energy structures (residues 3–163) of PagP in DPC micelles.

Figure 5

Topology model of PagP. Slowly exchanging backbone amides (exchange time constant >5 min) are shown with black lines, and HN-HN NOE connectivities are shown in red. Dashed lines indicate observations made for PagP-β-OG but not PagP-DPC, due to exchange broadening in the latter case. Residues 13–19, 64, 92, 96, 98, and 101 exhibit slow hydrogen exchange as well. Residues in squares are part of β-strands. Secondary structure is based on hydrogen bonding. Residues in yellow squares have side chains facing the membrane bilayer, whereas white squares indicate side chains lining the interior of the β-barrel. The presence of the β-bulge with the extension of strand A to Trp-32 is based on NOE data from PagP-β-OG; residues 31–33 and 152–153 are broadened beyond detection in PagP-DPC. The three residues important for catalytic activity as determined by site-directed mutagenesis, His-33, Asp-76, and Ser-77, are shown in blue.

Figure 6

(A) Heteronuclear ¹H-¹⁵N NOE, ¹⁵N T₁, and ¹⁵N T₂ values for PagP-DPC measured at 800 MHz plotted against PagP sequence and secondary structure. The relaxation measurements performed on PagP-β-OG displayed similar trends. (B) Ribbon representation of PagP in DPC. Residues colored in red have backbone ¹⁵N T₁s that are less than 80% of the value predicted for a 20-ns overall correlation time, S² = 0.85, τ_e = 10 ps (2.3 s). Yellow residues have NMR signals too faint to be detected in ¹H-¹⁵N HSQC spectra, and green residues have a weak but observable signal. All other residues are shown in blue.