NMR structure of r ALF‐ 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 r ALF‐ Pm3 protein. Its 3D structure was established by NMR and consists of three α‐helices packed against a four‐stranded β‐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 r ALF‐ Pm3 binds to LPS, lipid A and to OM®‐174, a soluble analogue of lipid A. Biophysical studies of r ALF‐ Pm3 /LPS and r ALF‐ 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 β‐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 β‐hairpin synthetic peptides corresponding to ALF‐L β‐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. © 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