Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry

Abstract

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type 11' 0-turn mimic of predetermined three-dimensional structure.The mimic &turn replaced residues GlyI6,l7 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' 0-turns in the native, unliganded enzyme. The 0-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the GlyI67l7-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the @turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid 0-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.

Evaluating β-turn mimics as β-sheet folding nucleators

“…Compound 5, designed to replace the i ϩ 1 and i ϩ 2 residues of a type II ␤-turn (25), was expected to introduce a Pin WW twist preference mismatch. Although 1 has been incorporated into folded proteins (40,41), its influence on folding kinetics is unknown. ␤-Turn mimics 4 and 5 have been included in place of loop 1 residues in Pin WW as part of a study to discern the energetic requirements for the transition from two-state folding to downhill folding (38).…”

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis.

“…Comparison of the substrate specificity of backbone-engineered HIV-1 PR with that of "native amide" enzymet was assessed by the cleavage of two synthetic peptides spanning the p17/p24 and p24/pl5 cleavage sites in the viral gag-pol protein (22). The p17/p24 peptide (500 ,uM) was incubated with backbone-engineered HIV-1 PR (35 nM) at 37°C in a pH 5.5 assay buffer containing 50 mM HOAc, 50 mM Mes, 100 mM Tris, 10% glycerol, and bovine serum albumin at 0.5 mg/ml, with the ionic strength adjusted to 1.0 by the addition of NaCl.…”

Synthesis of Native Proteins by Chemical Ligation

“…An early example of the utility of this approach was the total chemical synthesis of (BTD) HIV-1 protease (87), a protein in which the Gly-Gly sequence found in a β-turn in the native protein (20) was replaced by the sterically constrained bicyclic compound BTD, a rigid mimetic of type II β-turn geometry (88). The resulting enzyme showed full activity and a significantly enhanced thermostability (87).…”