Figure 1
Structure of 10E8ep peptide and energetics of binding to Fab 10E8 in membrane mimetics.
(a) Structural elements of gp41. The ectodomain pre-fusion structure of gp41 (PDB accession code: 4TVP) is depicted in a ribbon and tube representation above the diagram illustrating the relative positions of the most important constituents in the sequence: FP, fusion peptide; HR1 and HR2, amino- and carboxy-terminal heptad-repeat regions, respectively (helical ribbons), Loop, the region between HR1 and HR2 bearing a short, disulfide-bridged loop; MPER, membrane-proximal external region; TMD transmembrane domain. The ribbon structure below the diagram was modeled by superposing the NMR structures of the MPERp and CpreTM peptides solved in DPC micelles (PDB accession codes 2M8O and 2MG3, respectively), and supports the existence of two distinct helical segments: H1 and H2. (b) Env sequence covered by the 10E8ep peptide. (c) Structures adopted by 10E8ep in 25% (v/v) HFIP or 20 mM DPC. Left: The 20 lowest target function conformers overlaid onto the backbone atoms of residues 675-691. Lateral side-chain of Asp664, Trp672 and Lys683 residues are depicted in red, green and yellow, respectively. Right: Superposition of representative NMR structures for 10E8p in HFIP and in DPC displayed in ribbon representation. (d) Binding isotherm of the 10E8ep peptide to Fab 10E8 as measured by ITC. The upper panel indicates the heat released upon consecutive injections of 10 μL of peptide solution (40 μM) into Fab solution (3 μM) in the calorimeter cell; the lower panel depicts the integrated heats (symbols) and non-linear least-squares fit (line) of the data to a one-site binding model using ORIGIN 7.0. The thermodynamic parameters of binding are listed in Table 1. (e) Thermal stability of the Fab-peptide complex by DSC. DSC thermograms (dotted lines) for 10E8 Fab before (black) and after titration with peptide (blue) are shown. Unfolding of the Fab shows a single thermal transition in both cases. The midpoints of the thermal unfolding (TM) and the associated enthalpies (ΔH) are given. The values of TM and ΔH were obtained by curve fitting using ORIGIN 7.0 (solid lines).
