NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: Model of the possible lipid A-binding site.

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 (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(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.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.
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Yinshan Yang, Hélène Boze, Patrick Chemardin, André Padilla, Guy Moulin, et al.. NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: Model of the possible lipid A-binding site.. Biopolymers / Biopolymers (Biospectroscopy); Biopolymers (Pept Sci ); Biopolymers (Peptide Sci ); Biopolymers (Pept Sci); Biopolymers, 2009, 91 (3), pp.207-20. ⟨10.1002/bip.21119⟩. ⟨hal-00358510⟩



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