Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL from Pseudomonas aeruginosa.
Pathogenic gram-negative bacteria can modify the lipid A portion of their lipopolysaccharide in response to environmental stimuli. 3-O-deacylation of lipid A by the outer membrane enzyme PagL modulates signaling through Toll-like receptor 4, leading to a reduced host immune response. We found that PagL is widely disseminated among gram-negative bacteria. Only four residues are conserved: a Ser, His, Phe, and Asn residue. Here, we describe the crystal structure of PagL from Pseudomonas aeruginosa to 2.0-A resolution. It consists of an eight-stranded beta-barrel with the axis tilted by approximately 30 degrees with respect to the lipid bilayer. The structure reveals that PagL contains an active site with a Ser-His-Glu catalytic triad and an oxyanion hole that comprises the conserved Asn. The importance of active site residues was confirmed in mutagenesis studies. Although PagL is most likely active as a monomer, its active site architecture shows high resemblance to that of the dimeric 12-stranded outer membrane phospholipase A. Modeling of the substrate lipid X onto the active site reveals that the 3-O-acyl chain is accommodated in a hydrophobic groove perpendicular to the membrane plane. In addition, an aspartate makes a hydrogen bond with the hydroxyl group of the 3-O-acyl chain, probably providing specificity of PagL toward lipid A.
Rutten
Lucy
L
Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht, The Netherlands.
Geurtsen
Jeroen
J
Lambert
Wietske
W
Smolenaers
Jeroen J M
JJ
Bonvin
Alexandre M
AM
de Haan
Alex
A
van der Ley
Peter
P
Egmond
Maarten R
MR
Gros
Piet
P
Tommassen
Jan
J
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008050:Lipid A / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D008050:Lipid A; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases