Total chemical synthesis of human T-cell leukemia virus type 1 protease via native chemical ligation

Abstract

Human T-cell leukemia virus 1 (HTLV-1) protease, a member of the aspartic acid protease family, plays critical roles in the pathogenesis of the virus and is an attractive viral target for therapeutic intervention. HTLV-1 protease consists of 125 amino acid residues and functions as a homodimer stabilized in part by a four-stranded beta-sheet comprising the N- and C-termini. Compared with many other viral proteases such as HIV-1 protease, HTLV-1 protease is elongated by an extra 10 amino acid residue "tail" at the C-terminus. The structural and functional role of the extra C-terminal residues in the catalysis of HTLV-1 protease has been a subject of debate for years. Using the native chemical ligation technique pioneered by Kent and coworkers, we chemically synthesized a full-length HTLV protease and a C-terminally truncated form encompassing residues 1-116. Enzyme kinetic analysis using three different peptide substrates indicated that truncation of the C-terminal tail lowered the turnover number of the viral enzyme by a factor of 2 and its catalytic efficiency by roughly 10-fold. Our findings differ from the two extreme views that the C-terminal tail of HTLV-1 protease is either fully dispensable or totally required for enzyme dimerization and/or catalysis.

Figure 1

Strategy for the synthesis of HTLV-1 PR using three-segment native chemical ligation. The image of dimeric HTLV-1 PR was created by PyMol (DeLano Scientific LLC, http://pymol.org) using the coordinates for a truncated recombinant form (1–116) (PDB code: 2B7F) . The two ligation sites (site 1: Ser⁸⁹-Cys⁹⁰; site 2: Ser⁴⁶-Cys⁴⁷) and the catalytic residue Asp32 are highlighted in stick representation.

Figure 2

Characterization of synthetic HTLV-1 PR (1–126) and HTLV-1 PR (1–116) by analytical HPLC and ESI-MS. The chromatograms were obtained on a Waters symmetry 300 C₁₈ column (4.6×150 mm, 5 µM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min at 40 °C.

Figure 3

Cleavage of MA/^nitroCA by HTLV-1 PR (1–126) at different enzyme/substrate ratios. (A) [Enzyme] = 850 nM; [substrate] = 300 µM. (B) [Enzyme] = 1.25 µM; [substrate] = 200 µM. The cleavage reaction proceeded at 37 °C (in 250 mM phosphate, 5% glycerol, 1 mM EDTA, 5 mM DTT, 2 M NaCl, pH 5.6) for 1 h before being terminated by TFA acidification.

Figure 4

Enzyme kinetic measurements of the catalytic activity of HTLV-1 PR (1–126) (left column) and HTLV PR (1–116) (right column) with the substrates MA/CA (A, B), MA/^nitroCA (C, D), and CA/NC (E, F). The curves are averages of two independent measurements.

Figure 5

Apparent racemization at Ser⁸⁹. (A) Ligation of H-(47–89)αCOSR to H-(90-126)-OH. The chromatogram was obtained at a linear gradient of 20–50% acetonitrile containing 0.1% TFA under otherwise the same conditions as described in the legend of Figure 2. (B) Ligation of PIVLT^LSαCOSR to CLVDTK. (C) Ligation of PIVLT^DSαCOSR to CLVDTK. The ligation reactions of the model peptides were monitored at 40 °C on a Waters Xbridge™ C₁₈ column (3.5 µm, 4.6 × 150 mm) running a 30-min linear gradient of 5–65% acetonitrile containing 0.1% TFA at a flow rate of 1 ml/min. (D) Enzyme kinetic measurements of the catalytic activity of (^LS89→^DS89)-HTLV-1 PR (1–126) with the substrate CA/NC. The curves are averages of two independent measurements.