Importance of the N terminus of rous sarcoma virus protease for structure and enzymatic function

All retrovirus proteases (PRs) are homodimers, and dimerization is essentia l for enzymatic function. The dimer is held together largely by a short fou r-stranded antiparallel beta sheet composed of the four or five N-terminal amino acid residues and a similar stretch of residues from the C terminus. We have found that the enzymatic and structural properties of Rous sarcoma virus (RSV) PR are exquisitely sensitive to mutations at the N terminus. De letion of one or three residues, addition of one residue, or substitution o f alanine for the N-terminal leucine reduced enzymatic activity on peptide and protein substrates 100- to 1,000-fold. The purified mutant proteins rem ained monomeric up to a concentration of about 2 mg/ml, as determined by dy namic light scattering. At higher concentrations, dimerization was observed , but the dimer lacked or was deficient in enzymatic activity and thus was inferred to be structurally distinct from a wild-type dimer. The mutant pro tein lacking three N-terminal residues (Delta LAM), a form of PR occurring naturally in virions, was examined by nuclear magnetic resonance spectrosco py and found to be folded at concentrations where it was monomeric. This re sult stands in contrast to the report that a similarly engineered monomeric PR of human immunodeficiency virus type 1 is unstructured. Heteronuclear s ingle quantum coherence spectra of the mutant at concentrations where eithe r monomers or dimers prevail were nearly identical. However, these spectra differed from that of the dimeric wild-type RSV PR. These results imply tha t the chemical environment of many of the amide protons differed and thus t hat the three-dimensional structure of the Delta LAM PR mutant is different from that of the wild-type PR. The structure of this mutant protein may se rve as a model for the structure of the PR domain of the Gag polyprotein an d may thus give clues to the initiation of proteolytic maturation in retrov iruses.