ASSEMBLY OF A ROD-SHAPED CHIMERA OF A TRIMERIC GCN4 ZIPPER AND THE HIV-1 GP41 ECTODOMAIN EXPRESSED IN ESCHERICHIA-COLI

The HIV-1 envelope subunit gp41 plays a role in viral entry by initiat ing fusion of the viral and cellular membranes. A chimeric molecule wa s constructed centered on the ectodomain of gp41 without the fusion pe ptide, with a trimeric isoleucine zipper derived from GCN4 (pIIGCN4) o n the N terminus and part of the trimeric coiled coil of the influenza virus hemagglutinin (HA) HA2 on the C terminus. The chimera pII-41-HA was overexpressed as inclusion bodies in bacteria and refolded to sol uble aggregates that became monodisperse after treatment with protease , Either trypsin or proteinase K, used previously to define a protease -resistant core of recombinant gp41 [Lu, M., Blacklow, S. C. & Kim, P. S. (1995) Nat. Struct. Biol. 2, 1075-1082], removed about 20-30 resid ues from the center of gp41 and all or most of the HA2 segment, Eviden ce is presented that the resulting soluble chimera, retaining the pIIG CN4 coiled coil at the N terminus, is an oligomeric highly alpha-helic al rod about 130 Angstrom long that crystallizes. The chimeric molecul e is recognized by the Fab fragments of mAbs specific for folded gp41, A similar chimera was assembled from the two halves of the molecule e xpressed separately in different bacteria and refolded together. Cryst als from the smallest chimera diffract x-rays to 2.6-Angstrom resoluti on.