HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) complex (gp120/gp41). Binding of gp120 to receptors on the target cell triggers a series of conformational changes in the native Env trimer that ultimately lead to formation of a six-helix bundle structure in gp41, the so-called fusogenic state of the protein. In this state, interactions between two helical regions (HR1 and HR2) in the gp41 ectodomain are coupled to membrane fusion. HR2-derived peptides, such as Fuzeon (T20), are thought to inhibit membrane fusion by preventing the HR regions from refolding into the six-helix bundle. Despite intensive efforts, high-resolution structural information about the native, trimeric Env complex is lacking. Recognizing this history, we propose to apply new protein dissection approaches to study the pre-fusion conformation(s) of gp41. The broad, longterm objective of this proposal is to define the structural transitions of gp41 by which the HR regions, initially sequestered in the native Env complex, are released and refolded in order to cause membrane fusion. The specific aims of the proposal are: (1) To determine the effects of the fusion inhibitor T20-resistant/dependent mutations in the HR regions on the equilibrium folding, thermodynamics, and conformation of the six-helix bundle, in order to understand the mechanism of action of T20 and how its antiviral effect is mitigated by the evolution of resistant viruses in vivo. (2) To characterize, at high resolution, the structural and dynamic properties of the conserved membraneproximal Trp-rich domain of gp41 in association with [3OG micelles and lipid vesicles, in order to provide structural insight into the mechanism of HIV-1 neutralization by mAbs 2F5 and 4E10. (3) To crystallize and determine the structure of a novel trimerization domain formed by the HR2 and Trp-rich membrane-proximal regions, and to utilize engineered envelope glycoproteins to define the roles of this trimeric structural domain in regulating the stabilization and controlled activation of the native Env complex. We wish to elucidate a mechanism for the precise switch between the metastable native and fusogenic forms of the protein.