Crystal structure and catalytic mechanism of the LPS 3-<i>O</i>-deacylase PagL from<i>Pseudomonas aeruginosa</i>

Rutten, Lucy; Geurtsen, Jeroen; Lambert, Wietske; Smolenaers, J.J.; Bonvin, Alexandre M. J. J.; Haan, Alex de; Ley, Peter van der; Egmond, Maarten R.; Gros, Piet; Tommassen, Jan

doi:10.1073/pnas.0509392103

Cited by 66 publications

(97 citation statements)

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“…Before analysis, WC pellet was washed with PBS, whereas CE and PL were resuspended in Hepes at pH 7.0 and harvested by ultracentrifugation using identical procedures. Both in CE preparations and reconstituted in PL, PagL exhibited similar heat modifiability, a property typical of the wellfolded protein (12,13) (Fig. 1C).…”

Section: Resultsmentioning

confidence: 80%

“…1C). To verify that PagL was correctly folded in vivo and in vitro, we monitored its LPS 3-O-deacylase activity in CE and PL preparations as described previously (12,13). In both cases, LPS was converted into a form with higher electrophoretic mobility (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Solid-state NMR experiments were performed at a regulated sample temperature of −2°C on a narrow-bore Bruker Avance 700 spectrometer equipped with a 3.2-mm triple-resonance ( 1 H, 13 C, 15 N) E free MAS probe. 13 C and 1 H resonances were calibrated using adamantane as an external reference. The upfield 13 C resonance and isotropic 1 H resonance of adamantane were set to 31.47 and 1.7 ppm, respectively, to allow for a direct comparison of the solid-state chemical shifts to solution-state NMR data.…”

Section: Methodsmentioning

confidence: 99%

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Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

182

220

View full text Add to dashboard Cite

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“…However, no other PagL homolog could be identified in the genome sequence (data not shown). We recently solved the crystal structure of the P. aeruginosa PagL homolog (48). That study revealed a close resemblance of the active site of PagL to that of the outer membrane phospholipase A.…”

Section: Discussionmentioning

confidence: 99%

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

Geurtsen¹,

Steeghs

Hamstra³

et al. 2006

Infect Immun

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Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.

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“…The active sites of EptA and ArnT are located on the outer surface of the inner membrane (12). Activation of the PhoP/PhoQ two-component system by growth of cells at low divalent cation concentrations (13-15), or in the presence of cationic antimicrobial peptides (16), induces the genes encoding the outer membrane enzymes and PagL (20,21), which remodel the acyl chains of lipid A (Fig. 1).…”

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confidence: 99%

An Inner Membrane Dioxygenase that Generates the 2-Hydroxymyristate Moiety of Salmonella Lipid A

et al. 2008

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The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2 -dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A (Gibbons, H. S., Lin, S., Cotter, R. J., and Raetz, C. R. H. J. Biol. Chem. 275, 32940-49, 2000). We now demonstrate that inactivation of lpxO, which encodes a putative Fe 2+ /O 2 /α-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2 -[4′-32 P]-lipid A in vitro in the presence of Fe 2+ , O 2 , α-ketoglutarate, ascorbate and Triton X-100. The Fe 2+ chelator 2,2′-bipyridyl inhibits the reaction. The product generated in vitro is a mono-hydroxylated Kdo 2 -lipid A derivative. The [4′-32 P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from 32 P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3′-secondary myristoyl chain of Kdo 2 -lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe 2+ /O 2 /α-ketoglutarate-dependent dioxygenase family.The Salmonella typhimurium genome encodes several enzymes ( Fig. 1) that catalyze the covalent modification of the Kdo 2 -lipid A region of lipopolysaccharide (LPS) 1 (1,2). Activation of the PmrA/PmrB two component system by growth at low pH or as the result of point mutations in induces the transcription of the enzymes EptA and ArnT, which attach phosphoethanolamine (pEtN) 1 and 4-amino-4-deoxy-L-arabinose (L-Ara4N) 1 units to lipid A, respectively ( Fig. 1) (9-11). The active sites of EptA and ArnT are located on the outer surface of the inner membrane (12). Activation of the PhoP/PhoQ two-component system by growth of cells at low divalent cation concentrations (13-15), or in the presence of cationic antimicrobial peptides (16), induces the genes encoding the outer membrane enzymes and PagL (20,21), which remodel the acyl chains of lipid A (Fig. 1). The addition of a pEtN unit to the outer Kdo residue by EptB (Fig. 1) is independent of PmrA/PmrB and PhoP/ PhoQ, but instead is induced by 5 to 50 mM Ca 2+ ions (22,23).*Author to whom correspondence should be addressed: C. R. H. Raetz at (919) Fax (919) [33][34][35]. The active sites of these enzymes contain a single ferrous ion, usually coordinated by a His-X-Asp/Glu-X n -His facial triad motif in which n denotes...

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Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL fromPseudomonas aeruginosa

Cited by 66 publications

References 32 publications

Cellular solid-state nuclear magnetic resonance spectroscopy

Cellular solid-state nuclear magnetic resonance spectroscopy

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

An Inner Membrane Dioxygenase that Generates the 2-Hydroxymyristate Moiety of Salmonella Lipid A

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