NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.
The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs.
Yang
Yinshan
Y
CNRS UMR5048, INSERM, U554, Université Montpellier 1 et 2, Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, Cedex 9, France.
Boze
Hélène
H
Chemardin
Patrick
P
Padilla
André
A
Moulin
Guy
G
Tassanakajon
Anchalee
A
Pugnière
Martine
M
Roquet
Françoise
F
Destoumieux-Garzón
Delphine
D
Gueguen
Yannick
Y
Bachère
Evelyne
E
Aumelas
André
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003445:Crustacea / Q000737:chemistry*; D018360:Crystallography, X-Ray; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008050:Lipid A / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D020349:Surface Plasmon Resonance
D007447:Invertebrate Hormones; D008050:Lipid A