C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity

Abstract

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4–40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.

The comparative analysis of the protease molecule structure of the Human lymphotropic virus type-1 (HTLV-1)

“…By comparing the HTLV-1 and HIV-1 protease structure and sequence, the enzymatic digestion and substrate binding sites were identified. Despite the high similarity of these two proteins, HIV-1 protease inhibitors have not any effect on HTLV-1 protease (6). Regarding this fact, there are not any other studies about the differences between various HTLV-1 virus subtypes and this study was done for the first time.…”

“…HTLV-1 is capable to make long period of infection in individuals (4,5). Because of longterm and latent infection the symptoms of disease in about 5% of carrier (2,3,6) which appear as Adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) (1, 2, H 7,8). Nowadays, about 10-20 million people are infected with this virus worldwide (2,9,10).…”

Development of [Ile40]HTLV‐I protease inhibition assay using novel fluorogenic and chromogenic substrate

“…The [L40I,C90A,C109A]‐mutant protease was reported to exhibit little change in kinetic properties relative to the wild‐type HTLV‐I protease and are considered more stable because of lower risks for undesired intramolecular disulfide bond formations18, 20. In addition, removal of the last nine or ten C ‐terminal residues (116–125 or 117–125) does not drastically negatively impact the proteases' kinetic profiles16, 21, 22, despite a conflicting report23. Due to the intense self‐aggregation, the three‐dimensional structural data of several protein–inhibitor complexes could only be solved when the nine C ‐terminal residue was removed from the [Ile 40 ]HTLV‐I protease16, 17.…”

“…These reports suggest our L40I mutant protease should have similar catalytic efficiency as our His‐tagged non‐mutated protease. However, there are also huge discrepancies in the literature: the catalytic efficiencies of the wild‐type HTLV‐I protease and CA/NC substrate have been reported as 158.7 m M −1 s −1 in one research group23 and 0.019 m M −1 s −1 in another10. Another research group showed the inconsistency was a result of different refolding conditions22.…”

Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors

“…10 But recent research reports state that anti-HIV PR drugs cannot function as HTLV-1 PR blockers and several successful HIV-1 PR inhibitors failed to provide the inhibitory activity against HTLV-1 PR, 8,11 so prediction of a potent inhibitor for HTLV-1 PR is highly essential for human welfare. 13 The two chains of the HTLV-1 PR monomer are bound by nonbonding interactions with the active site at the interface between two monomers. 12 Structure of HTLV-1 PR represents the homodimer, containing 125 residues per chain, which is a longer sequence compared with HIV-1 PR.…”