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PMID: 9177169 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Assembly of a rod-shaped chimera of a trimeric GCN4 zipper and the HIV-1 gp41 ectodomain expressed in Escherichia coli.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 94 ·No. 12 ·1997-06-10 ·Pages 6065-9

Weissenhorn W, Calder LJ, Dessen A, Laue T, Skehel JJ, Wiley DC

Abstract

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.

MeSH Terms
Antibodies Cloning, Molecular Cross-Linking Reagents Crystallography, X-Ray DNA-Binding Proteins Escherichia coli Fungal Proteins/biosynthesis,chemistry Genes, env HIV Envelope Protein gp41/biosynthesis,chemistry HIV-1/metabolism Hemagglutinin Glycoproteins, Influenza Virus/biosynthesis,chemistry Macromolecular Substances Microscopy, Electron Protein Folding Protein Kinases/biosynthesis,chemistry Protein Structure, Secondary Recombinant Fusion Proteins/biosynthesis,chemistry,isolation & purification Saccharomyces cerevisiae Proteins Transcription Factors/biosynthesis,chemistry
Chemicals
Antibodies Cross-Linking Reagents DNA-Binding Proteins Fungal Proteins HIV Envelope Protein gp41 Hemagglutinin Glycoproteins, Influenza Virus Macromolecular Substances Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins Transcription Factors Protein Kinases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Weissenhorn W
Laboratory of Molecular Medicine, The Children's Hospital, 320 Longwood Avenue, Boston, MA 02215, USA.
Calder L J
Dessen A
Laue T
Skehel J J
Wiley D C
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.94.12.6065
Published
1997-06-10
Pages
6065-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC21001
Subset
IM
Grants
NIGMS NIH HHS · GM39589 · United States
External Links
PubMed source DOI: 10.1073/pnas.94.12.6065 PMC Full Text View journal details
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