All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.

• MeSH
• Algorithms MeSH
• Aspartic Acid Endopeptidases chemistry   genetics   metabolism   MeSH
• Point Mutation genetics   MeSH
• Dimerization MeSH
• Fluorescent Dyes metabolism   MeSH
• HIV Protease chemistry   genetics   metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Models, Molecular MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Retroviridae Proteins chemistry   genetics   metabolism   MeSH
• Serine chemistry   genetics   MeSH
• Enzyme Stability genetics   MeSH
• Substrate Specificity MeSH
• Threonine chemistry   genetics   MeSH
• Binding Sites genetics   MeSH
• Hydrogen Bonding MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Endopeptidases MeSH
• Fluorescent Dyes MeSH
• HIV Protease MeSH
• protease p15 MeSH Browser
• Recombinant Proteins MeSH
• Retroviridae Proteins MeSH
• Serine MeSH
• Threonine MeSH

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

• MeSH
• Dimerization MeSH
• HIV-1 enzymology   MeSH
• HIV Protease chemistry   genetics   metabolism   MeSH
• Hydrolysis MeSH
• Catalysis MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Mutagenesis, Site-Directed MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Substrate Specificity MeSH
• Threonine chemistry   genetics   metabolism   MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• HIV Protease MeSH
• Recombinant Proteins MeSH
• Threonine MeSH