Solid-State NMR Structural Measurements on the Membrane-Associated Influenza Fusion Protein Ectodomain
Jaime Curtis-Fisk, Casey Preston, Zhaoxiong Zheng, R. Mark Worden, and David P. Weliky*
The 185 residue "FHA2" domain of the influenza virus hemagglutinin protein lies outside the virus and plays a significant role in catalyzing fusion between viral and host cell membranes. FHA2 was expressed in Escherichia coli with a yield of 3 mg/L of fermentation and after purification was shown to be folded in detergent and to rapidly catalyze vesicle fusion. More than half of the FHA2 residues are the first residue in a unique sequential pair, and for a particular pair, 13CO and 15N amino acid type labeling was, respectively, done for the first and second residues in the pair. For membrane-associated FHA2, NMR filtering allowed selective observation of the 13CO signal of the first residue, and the 13CO chemical shift was correlated with local secondary structure. Such shift/conformational measurements were made at critical glycine residues in the functionally important "fusion peptide" region of FHA2 and at a more C-terminal leucine. This should be a general approach to mapping the conformation of membrane-associated FHA2 and for testing FHA2 structure-function models. The approach will also be applicable to other viral fusion proteins and other large membrane proteins.
J. Am. Chem. Soc., 129 (37), 11438 -11446, 2007.
Phosphate-Mediated Arginine Insertion into Lipid Membranes and Pore Formation by a Cationic Membrane Peptide from Solid-State NMR
Ming Tang, Alan J. Waring, and Mei Hong*
The insertion of charged amino acid residues into the hydrophobic part of lipid bilayers is energetically unfavorable yet found in many cationic membrane peptides and protein domains. To understand the mechanism of this translocation, we measured the 13C-31P distances for an Arg-rich -hairpin antimicrobial peptide, PG-1, in the lipid membrane using solid-state NMR. Four residues, including two Arg's, scattered through the peptide were chosen for the distance measurements. Surprisingly, all residues show short distances to the lipid 31P: 4.0-6.5 Å in anionic POPE/POPG membranes and 6.5-8.0 Å in zwitterionic POPC membranes. The shortest distance of 4.0 Å, found for a guanidinium C at the -turn, suggests N-H···O-P hydrogen bond formation. Torsion angle measurements of the two Arg's quantitatively confirm that the peptide adopts a -hairpin conformation in the lipid bilayer, and gel-phase 1H spin diffusion from water to the peptide indicates that PG-1 remains transmembrane in the gel phase of the membrane. For this transmembrane -hairpin peptide to have short 13C-31P distances for multiple residues in the molecule, some phosphate groups must be embedded in the hydrophobic part of the membrane, with the local 31P plane parallel to the -strand. This provides direct evidence for toroidal pores, where some lipid molecules change their orientation to merge the two monolayers. We propose that the driving force for this toroidal pore formation is guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane. This phosphate-mediated translocation of guanidinium ions may underlie the activity of other Arg-rich antimocrobial peptides and may be common among cationic membrane proteins.