Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: Rethinking the "fireman's grip" hypothesis

Aspartic proteinases share a conserved network of hydrogen bonds (termed "f ireman's grip"), which involves the hydroxyl groups of two threonine residu es in the active site Asp-Thr-Cly triplets (Thr26 in the case of human immu nodeficiency virus type 1 (HIV- I) PR). In the case of retroviral proteinas es (PRs), which are active as symmetrical homodimers, these interactions oc cur at the dimer interface. For a systematic analysis of the "fireman's gri p," Thr26 of HIV-I PR was changed to either Ser, Cys, or Ala. The variant e nzymes were tested for cleavage of HIV-I derived peptide and polyprotein su bstrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-I PR, indicating that the sulfhydryl group of cy steine can substitute for the hydroxyl of the conserved threonine in this p osition. PR(T26A), which lacks the "Fireman's grip" interaction, was virtua lly inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effe ct when introduced into only one chain of a linked dimer of HIV-I PR. In th is case, even changing both Thr residues to Ala yielded residual activity s uggesting that the "fireman's grip" is not essential for activity but contr ibutes significantly to dimer formation. Taken together, these results indi cate that the "fireman's grip" is crucial for stabilization of the retrovir al PR dimer and for overall stability of the enzyme.