Macromolecular inhibitors of HIV-1 protease. Characterization of designed heterodimers.

Defective variants of human immunodeficiency virus type 1 (HIV-1) protease (HIV PR) have been engineered to inhibit wild-type (wt) HIV PR activity. These variants were designed to promote the formation of heterodimers and to destabilize the formation of inactive variant homodimers of HIV-1 protease through substitutions at Asp-25, Ile-49, and Gly-50 (Babé, L. M., Rosé, J., and Craik, C. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10069-10073; McPhee, F., Good, A. C., Kuntz, I. D., and Craik, C. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11477-11481). The mechanism of action of these dominant-negative inhibitors was established using recombinantly expressed defective monomers. The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity. This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers. Heterodimer formation was detected by (i) isolating refolded, inactive heterodimers using histidine-tagged defective monomers and (ii) isolating heterodimers from bacteria coexpressing both wt and defective variants of HIV PR. Single-chain variants of HIV PR, in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyzed. Thermal denaturation of these single-chain heterodimers using differential scanning calorimetry revealed a 1.5-7.2 degrees C greater thermal stability than single-chain wt HIV PR. The thermodynamic trend shown by these three variants mirrors their relative inhibition in provirus transfection assays. These data support the model that the effects seen both in tissue culture and in vitro arise from an increase in stability conferred on these heterodimers by interface mutations and identifies heterodimer formation as their mechanism of inhibition.

Structural basis of the oligomerization of hepatitis delta antigen.

BACKGROUND: The hepatitis D virus (HDV) is a small satellite virus of hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe liver disease in humans. The small 195 amino-acid form of the hepatitis delta antigen (HDAg) functions as a trans activator of HDV replication. A larger form of the protein containing a 19 amino acid C-terminal extension inhibits viral replication. Both of these functions are mediated in part by a stretch of amino acids predicted to form a coiled coil (residues 13-48) that is common to both forms. It is believed that HDAg forms dimers and higher ordered structures through this coiled-coil region. RESULTS: The high-resolution crystal structure of a synthetic peptide corresponding to residues 12 to 60 of HDAg has been solved. The peptide forms an antiparallel coiled coil, with hydrophobic residues near the termini of each peptide forming an extensive hydrophobic core with residues C-terminal to the coiled-coil domain in the dimer protein. The structure shows how HDAg forms dimers, but also shows the dimers forming an octamer that forms a 50 A ring lined with basic sidechains. This is confirmed by cross-linking studies of full-length recombinant small HDAg. CONCLUSIONS: HDAg dimerizes through an antiparallel coiled coil. Dimers then associate further to form octamers through residues in the coiled-coil domain and residues C-terminal to this region. Our findings suggest that the structure of HDAg represents a previously unseen organization of a nucleocapsid protein and raise the possibility that the N terminus may play a role in binding the viral RNA.

Self-association of a synthetic peptide from the N terminus of the hepatitis delta virus protein into an immunoreactive alpha-helical multimer.

The formation of hepatitis delta antigen (HDAg) multimers is required for full biologic activity of this protein and for replication of the hepatitis delta virus. To determine the residues responsible for multimerization, three peptides [ delta 12-49, delta 25-60(Y), delta 12-60(Y)] from the putative coiled-coil multimer-forming domain of HDAg were chemically synthesized and biophysically characterized by circular dichroic spectroscopy, deuterium-exchange mass spectrometry, gel filtration, chemical crosslinking, and ultracentrifugation. By circular dichroism the 50-residue peptide delta 12-60(Y) was half-denatured above 80 degrees C and was 97% alpha-helical at 5 degrees C and 84% alpha-helical at 37 degrees C. By deuterium exchange, peptide delta 12-60(Y) was 93% alpha-helical at 25 degrees C. Its high alpha-helicity and melting temperature are due to the formation of an alpha-helical multimer consisting of four or more chains. All three synthetic peptides reacted with human anti-HDAg antibodies in an enzyme-linked immunosorbent assay, but only peptide delta 12-60(Y) was detected in a sandwich radioimmunoassay in which successful antigens must display at least two antibody-binding sites, which correlates with the ability of this peptide to form multimers. Peptide delta 12-60(Y) also interfered with the self-association of natural HDAg into multimers. These results have significant practical implications for development of improved diagnostic tests, antiviral agents, and possibly even vaccines for prevention of hepatitis delta virus disease.

Rational design of a three-heptad coiled-coil protein and comparison by molecular dynamics simulation with the GCN4 coiled coil: presence of interior three-center hydrogen bonds.

alpha-Helical coiled coils have a 7-residue repeating pattern (abcdefg) where a and d are usually hydrophobic. We have designed a 2-stranded 44-residue coiled-coil protein (P44) consisting of 2 22-residue alpha-helices linked by 2 terminal disulfide groups to test whether the disulfide bridges could stabilize a 3-heptad coiled coil. P44 should be stabilized by intrahelical hydrogen bonds, interhelical disulfide and salt bridges, and interior hydrophobic interactions. A computer model of P44 was built and its stability was studied by molecular dynamics simulation with explicit water. This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation with explicit water. This doubly crosslinked 3-heptad coiled coil did not unfold during a 300-ps simulation. But reduced P44 with 4 thiol groups did unfold. For comparison, the 62-residue crystal structure of the 4-heptad coiled coil of transcription activator GCN4 did not unfold during a 300-ps simulation. Thus P44 may be a stable folded protein in aqueous solution. These simulations revealed the presence of 2 local hydrogen bond networks involving intra-helical 3-center hydrogen bonds in the hydrophobic interior of the coiled coils of GCN4 and P44. The NH hydrogen at d makes a 3-center hydrogen bond whose major component is to the i - 4 C = O oxygen at g and minor component is to the solvent-inaccessible i - 3 C = O oxygen at a. Likewise, the NH hydrogen at g makes a 3-center hydrogen bond with the i - 4 C = O oxygen at c and the buried i - 3 C = O oxygen at d.