Crystallographic and biophysical analysis of a bacterial signal peptidase in complex with a lipopeptide-based inhibitor.
We report here the crystallographic and biophysical analysis of a soluble, catalytically active fragment of the Escherichia coli type I signal peptidase (SPase Delta2-75) in complex with arylomycin A2. The 2.5-A resolution structure revealed that the inhibitor is positioned with its COOH-terminal carboxylate oxygen (O45) within hydrogen bonding distance of all the functional groups in the catalytic center of the enzyme (Ser90 O-gamma, Lys145 N-zeta, and Ser88 O-gamma) and that it makes beta-sheet type interactions with the beta-strands that line each side of the binding site. Ligand binding studies, calorimetry, fluorescence spectroscopy, and stopped-flow kinetics were also used to analyze the binding mode of this unique non-covalently bound inhibitor. The crystal structure was solved in the space group P4(3)2(1)2. A detailed comparison is made to the previously published acyl-enzyme inhibitor complex structure (space group: P2(1)2(1)2) and the apo-enzyme structure (space group: P4(1)2(1)2). Together this work provides insights into the binding of pre-protein substrates to signal peptidase and will prove helpful in the development of novel antibiotics.
Paetzel
Mark
M
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6 Canada. mpaetze@sfu.ca
Goodall
Jonathon J
JJ
Kania
Malgosia
M
Dalbey
Ross E
RE
Page
Malcolm G P
MG
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008074:Lipoproteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D008024:Ligands; D008074:Lipoproteins; D008565:Membrane Proteins; D009842:Oligopeptides; C488532:arylomycin A2; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D010100:Oxygen