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 initiating fusion of the viral and cellular membranes. A chimeric molecule was constructed centered on the ectodomain of gp41 without the fusion peptide, with a trimeric isoleucine zipper derived from GCN4 (pIIGCN4) on 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 soluble 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 residues from the center of gp41 and all or most of the HA2 segment. Evidence is presented that the resulting soluble chimera, retaining the pIIGCN4 coiled coil at the N terminus, is an oligomeric highly alpha-helical rod about 130 A long that crystallizes. The chimeric molecule is recognized by the Fab fragments of mAbs specific for folded gp41. A similar chimera was assembled from the two halves of the molecule expressed separately in different bacteria and refolded together. Crystals from the smallest chimera diffract x-rays to 2.6-A resolution.
Weissenhorn
W
W
Laboratory of Molecular Medicine, The Children's Hospital, 320 Longwood Avenue, Boston, MA 02215, USA.
Calder
L J
LJ
Dessen
A
A
Laue
T
T
Skehel
J J
JJ
Wiley
D C
DC
D000906:Antibodies; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D015752:Genes, env; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis / Q000737:chemistry*; D015497:HIV-1 / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000737:chemistry*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D017510:Protein Folding; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry
D000906:Antibodies; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015700:HIV Envelope Protein gp41; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases