The ESI-MS spectra of PRWT and PRD25N revealed four peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,500. Since +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z=2164.51 as calculated with their average mass in the case of PRD25N), the greatest peak detected at m/z 2164.51 was determined to represent two forms, a PR monomer and PR dimer, thus being [PRD25N]5+ and [2PRD25N]10+. In the present study, we designatee a monomer and a dimer ion of PRX as [PRX]Y and [2PRX]Y, respectively, where X denotes an amino acid substitution(s) and Y denotes a charge of ion. To determine whether the detected ions represented monomers and/or dimers, we examined multiply-charged isotopologue clusters of PRD25N using the Solarix FT-ICR MS (Bruker Daltonics) and analyzed the difference in m/z ratios of two adjacent isotope peaks because monomer and dimer PRD25N ions with the same m/z show different peak values in order of their charges. We also constructed three mutated protease species containing amino acid substitutions at the active site (PRD29N, PRT26A, and PRR87K), a C-terminus-truncated mutant (PR1-C95A), and the mutant including L97A and F99A substitutions (PR97/99). Two PRD29N dimer ions ([2PRD29N]11+ and [2PRD29N]9+) were detected, while no dimer ions were detected with PRT26A and PRR87K. Additional analyses of the isotopologue ion peaks with PRT26A and PRR87K confirmed the absence of dimer ions. Importantly, two PR1-C95A dimer ions ([2PR1-C95A]11+ and [2PR1-C95A]9+) were identified although PR1-C95A monomer ion ([PR1-C95A]6+) was found to be a major peak. Two PR97/99 dimer ions were also detected. Considering that [PRWT]5++[2PRWT]10+ representing monomers+dimers was found to be a major peak together with a minor peak of [PRWT]6+, the PR1-C95A and PR97/99 species had a significantly reduced but persistent ability to dimerize in comparison to PRWT. Taken together, the data strongly suggest that the protease dimerization process consists of two distinct steps: (i) initial albeit weak intermolecular interactions occurring in the active site interface, constructing unstable or transient dimers and (ii) subsequent interactions in the termini interface, resulting in the complete and tighter protease dimerization. To regard the thermal stability of PRWT and various mutated species, we employed the differential scanning fluorimetry (DSF). The order of thermal stability was PRWT PRD25N PRD29N PR97/99 PRT26A PRR87K PR1-C95A (Tm;53.37 52.18 51.02 48.22 48.12 47.02 44.46 C, respectively). The difference in Tm values between PRD25N and PRD29N (1.16 C) was less than the difference between PRD25N and PR1-C95A (7.62 C), indicating that in terms of thermal stability, PRD25N is closer to PRD29N compared to the most unstable PR1-C95A. Thus, PRD29N monomer subunits are likely to interact at the active site interface and subsequently at the termini interface, forming stable dimers. The DSF data, however, showed that Tm value of PR97/99 (48.22 C) was quite low compared to that of PRWT (53.37 C) and PRD29N (51.02 C), suggesting that PR97/99 dimers are likely to be unstable. The Tm value of PR1-C95A was further lower (44.46 C), suggesting that PR1-C95A dimers are also likely to be unstable. Taken the ESI-MS and DSF results together, one can say that the present ESI-MS assay detects both unstable (transient) and stable dimers. Furthermore, DSF data indicated that the stability of PR97/99 and PR1-C95A dimers were lower than that of PRD25N dimer (dimer dissociation constant;KD=1.3 microM), suggesting KD value of unstable or transient dimers were higher than 1.3 microM (21). We previously reported that DRV unhibit not only proteolytic activity but also PR dimerization, while two FDA-approved anti-HIV-1 drugs, saquinaqvir (SQV) and nelfinavir (NFV), showed no dimerization inhibition activity as examined with the FRET-based HIV-1 expression system. To analyze the mechanism of the PR dimerization inhibition by DRV, we therefore examined the binding properties of DRV, SQV and NFV with PRWT. The ESI-MS spectrum of PRWT without drugs showed four peaks derived from differently charged ions, [PRWT]6+, [2PRWT]11+, [PRWT]5++[2PRWT]10+, and [2PRWT]9+. In the presence of DRV, four additional peaks appeared, ([PRWT+DRV]6+, [2PRWT+DRV]10+, [PRWT+DRV]5+, and [2PRWT+DRV]9+). Additional analysis of the isotopologue ion peaks with PRD25N in the presence of DRV confirmed the identity of monomer and dimer ions. On the other hand, the binding of SQV to PRWT yielded only two additional peaks, [2PRWT+SQV]10+ and [2PRWT+SQV]9+, indicating that SQV binds only to PRWT dimers, not to monomers. In the presence of NFV, as in the case of SQV, two additional peaks, [2PRWT+NFV]10+ and [2PRWT+NFV]9+, were identified. The relatively weak intensity of SQV- and NFV-bound PRWT dimers is presumably due to their relatively low binding affinity to PRWT. Taken together, these data clearly indicate that SQV and NFV bind to PRWT dimers but not to monomers and DRV inhibits PR dimerization by binding to PR monomers in a one-to-one molar ratio. Highly DRV-resistant HIV-1 isolates we generated in vitro had acquired a unique combination of 4 amino acid substitutions (V32I/L33F/I54M/I84V) and DRV had decreased its binding to PR monomers containing such 4 amino acid substitutions. We, therefore, examined whether such 4 amino acid substitutions altered the binding profiles of DRV with PR using ESI-MS. The ESI-MS spectrum of PR32/33/54/84 re-folded in the absence of DRV showed four peaks derived from 5 differently charged ions [PR32/33/54/84]6+, [2PR32/33/54/84]11+, [PR32/33/54/84]5++[2PR32/33/54/84]10+, and [2PR32/33/54/84]9+. However, in the presence of DRV, only a substantially low peak representing DRV-bound PR32/33/54/84 dimers or [2PR32/33/54/84+DRV]10+ was detected at m/z 2230.05 and no DRV-bound PR monomers were detected. Thus, it seems that the loss of binding affinity to PR32/33/54/84 in the monomeric form is greater than that in the dimeric form.. Finally, we asked if DRV had an ability to bind to the PR precursor protein, Gag-Pol polyprotein, which is produced through the frameshifting process in the Gag-encoding gene translation and subsequently maturates following the duly excision through autoproteolysis. To examine the DRV binding to the PR precursor protein, a transframe precursor form of PR containing D25N substitution, TFR-PRD25N, was constructed. In the absence of drugs, TFR-PRD25N generated [TFR-PRD25N]10+, [TFR-PRD25N]9+, [TFR-PRD25N]8+, and [TFR-PRD25N]7+, indicating that TFR-PRD25N failed to dimerize, in line with the NMR data reported by Ishima and her colleagues. In the presence of DRV, two additional peaks [TFR-PRD25N+DRV]8+ and [TFR-PRD25N+DRV]9+ appeared, indicating that DRV bound to the TFR-PRD25N monomers. It has been shown that the addition of C-terminus 4 AAs (PISP) to TFR-PR increases thermal stability of DRV-bound TFR-PR. In the present study, we generated TFR-PRD25N-7AA, which contained additional seven N-terminus AAs of reverse transcriptase (7AA;PISPIET) at the C-terminus of TFR-PRD25N. The ESI-MS revealed that TFR-PRD25N-7AA formed dimers, suggesting that the addition of the seven AAs allowed TFR-PRD25N-7AA to dimerize probably by giving TFR-PRD25N-7AA proper conformation. The ESI-MS then showed that DRV binds to both TFR-PRD25N-7AA monomers and dimmers. These results strongly suggest that the loss of dimerization ability of TFR-PRD25N resulted in the loss of DRV's dimer binding. The present data set should represent the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV.
Showing the most recent 10 out of 31 publications
