Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.
Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.
Schutkowski
M
M
Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Arbeitsgruppe 'Enzymologie der Peptidbindung', Halle/Saale, Germany.
Drewello
M
M
Wöllner
S
S
Jakob
M
M
Reimer
U
U
Scherer
G
G
Schierhorn
A
A
Fischer
G
G
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D002352:Carrier Proteins; D015683:Gene Products, gag; D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase