Abstract
The serine protease trypsin forms a tightly bound inhibitor complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with the negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to alpha-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before and unrestrained Umbrella Sampling simulations of these complexes suggest additional energetic minima. Here, we use Random Acceleration Molecular Dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the pre-bound state. The pre-bound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the the BPTI variants and change in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between fully bound state and pre-bound state and also lowers the energy minimum of the pre-bound state. While the effect of fluorination is in general difficult to quantify, here it is in part caused by a favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the pre-bound state offers insight into the inhibitory mechanism of BPTI and might add valuable information for the design serine protease inhibitors.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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