Thermodynamics of trimer-of-hairpins formation by the SIV gp41 envelope protein

The gp41 envelope protein mediates the entry of primate immunodeficiency vi ruses into target cells by promoting the fusion of viral and cellular membr anes. The structure of the gp41 ectodomain core represents a trimer of iden tical helical hairpins in which a central trimeric coiled-coil made up of t hree amino-terminal helices is wrapped in an outer layer of three antiparal lel carboxyl-terminal helices. Triggering formation of this fusion-active g p41 conformation appears to cause close membrane apposition and thus overco me the activation energy barrier for lipid bilayer fusion. We present a det ailed description of the folding thermodynamics of the simian immunodeficie ncy virus (SIV) gp41 core by using a recombinant trimeric N34(L6)C28 model. Differential scanning calorimetry and spectroscopic experiments on denatur ant-induced and thermal unfolding indicate that the free energy of associat ion of three 68 residue N34(L6)C28 peptides to a trimer-of-hairpins is 76 k J mol(-1) at pH 7.0 and 25 degreesC in physiological buffer. The associated enthalpy change, DeltaH(unf), is 177 kJ mol(-1), while the entropy of unfo lding, DeltaS(unf), is 0.32 kJ K-1 mol(-1). The temperature of maximal stab ility is close to 20 degreesC. The unfolding heat capacity increment is sim ilar to9 kJ K-1 mol(-1) (similar to 45 J K-1 mol residue(-1)), which is low er than expected for unfolding of the trimer to an extended and fully hydra ted polypeptide chain. Replacement by isoleucine of the polar residues Thr5 82 or Thr586 buried in the N-terminal trimeric coiled-coil interface leads to very strong stabilization of the trimer-of-hairpins, 30-35 kJ mol(-1). S ingle-point mutations in the central coiled-coil thus strongly stabilize th e gp41 core structure. These thermodynamic characteristics may be important for the refolding of the gp41 envelope protein into its fusion-active conf ormation during membrane fusion. (C) 2001 Academic Press.