NMR Structure of rALF-Pm3, an Anti-Lipopolysaccharide Factor from Shrimp: Model of the Possible Lipid A-Binding Site
|Author(s)||Yang Yinshan1, Boze Helene2, Chemardin Patrick2, Padilla Andre1, Moulin Guy2, Tassanakajon Anchalee3, Pugniere Martine4, Roquet Francoise4, Destoumieux Garzon Delphine5, Gueguen Yannick5, Bachere Evelyne5, Aumelas Andre1|
|Affiliation(s)||1 : Univ Montpellier 1 & 2, INSERM, CNRS,U554, Ctr Biochim Struct,UMR5048, F-34090 Montpellier 9, France.
2 : Genie Microbiol & Enzymat, UMR SPO INRA, F-34060 Montpellier, France.
3 : Chulalongkorn Univ, Fac Sci, Dept Biochem, Shrimp Mol Biol & Genom Lab, Bangkok 10330, Thailand.
4 : Univ Montpellier 1, CPBS, F-34095 Montpellier, France.
5 : Univ Montpellier 2, CNRS, UMR Ecosyst Lagunaires 5119, IFREMER, F-34095 Montpellier 5, France.
|Source||Biopolymers (0006-3525) (Wiley / Blackwell), 2009-03 , Vol. 91 , N. 3 , P. 207-220|
|WOS© Times Cited||63|
|Keyword(s)||Ultracentrifugation, Surface plasmon resonance, Septic shock, Structure, NMR, Lipid A, Lipopolysaccharide, Anti lipopolysaccharide factor|
|Abstract||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 N-15 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(R)-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM(R)-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. (C) 2008 Wiley Periodicals, Inc. Biopolymers 91: 207-220, 2009.|