Trans-Membrane Domain of HIV gp41 Interacts with the Externally Added gp41 Fusion Peptide: TMD-FP Complex Inhibits Model Membrane Fusion

Abstract

We report the effects of a complex between fusion peptide (FP) and trans-membrane domain (TMD) of gp41, that separately promote fusion, on the kinetics of PEG-mediated fusion and on the structure of the bilayer. Circular dichroism (CD) measurements showed that FP in the membrane exists mostly in a beta sheet at L/P ratios 1200/1 to 200/1, while the gp41 TMD displayed ∼32% helix, 23% beta, 21% turn and 24% other secondary structure content at L/P of 300/1. Adding FP to membranes containing TMD resulted in a TMD-FP complex with reduced helical content and increased beta and turn content. DPH and TMA-DPH anisotropy measurements revealed that the FP-TMD complex reduced membrane order in both the interface and interior regions of the bilayer, whereas FP alone increased order in both regions. The complex also increased water penetration into the bilayer (lifetime ratio of TMA-DPH in H2O and D2O) much more than for the individual peptides. While the FP decreased membrane free volume (from partitioning of C6NBDPC from micellar to bilayer phase), the presence of TMD enhanced this effect enormously. The time courses of lipid mixing (LM), content mixing (CM) and content leakage (L) were fitted globally to 3-state or 4-state sequential models (Biophys. J., 2007, 92; 4012), providing estimates of rate constants for inter-conversion between states as well as probabilities of the occurrence of LM, CM or L in each state. The FP-TMD complex inhibited the rates of initial intermediate but especially pore formation. The effect on pore formation was predominantly due to a large increase in the activation enthalpy not matched by as large an increase activation entropy. Supported by NIGMS grant 32707 to BRL.