Previous studies have suggested that, before their opening, the flaps separate widely up to 30 Å [19,27]. Despite the discrepancy of the exact extent of opening, both these reported studies [19,27] and our study suggested that the flaps may separate wide enough to allow the entry and subsequent binding of the substrates or inhibitors to the catalytic center. In the flaps of the inhibitor-free HIV-1 protease, the flexible region is mostly localized in the tips, especially in the early stage of opening process (Fig. 3). In the first 2.0 ns, the RSMD of the backbone Cα of the HIV-1 protease and that of the tips (residues from 43 to 58) are about 2.0 Å; whereas after 2.0 ns, that of the β-hairpins increases to 4.0 Å sharply. This is in agreement with the previous study that has shown the β-hairpins are present in solution and are well ordered except at the tips [13,16]. We found that the flap β-hairpins each moves as a rigid body with highly flexible tips, with the flap elbows acting as ‘hinges’, which is consistent with the results of Hornak et al. . However, the ‘rigid body’ also has some soft characters and has some ‘fluctuating-like’ movements during the simulation. But our simulation showed that when the β-hairpins have separated up to about 10 Å, the HIV-1 protease is still at semi-open form by the metric suggested by Hornak . Other regions of the HIV-1 protease are less structurally variable. For instance, the conformations of the C-termini and N-termini ends were largely constrained by the intact hydrogen bonds during the simulation.
Our simulation study reveals the existence of an alternative opening path to the semi-open conformation of HIV-1 protease with respect to those exposed by earlier simulation studies. Our results are consistent with the opening behavior detected by some experimental studies that is otherwise un-explainable by the opening paths of the earlier simulation studies . Via this alternative path, the highly flexible flap tips are ready to open to such an extent that they allow the entrance and binding of substrates and inhibitors. Our study combined with earlier studies suggests the existence of multiple opening paths and molecular dynamics simulation is capable of identifying these paths and in a broader perspective the multiple transition paths between different states of proteins.
The present study is supported by the National Natural Science Foundation of China (No. 20873087).
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