Nature of irreversible inhibition of human 20S proteasome by Salinosporamide A. The critical role of Lys-Asp dyad revealed from electrostatic effects analysis

Abstract

20S proteasome is the enzymatic core engine involved in the processes of cellular protein regulation, thus becoming a promising target inter alia in the therapy of many diseases. In this work, the mechanism of the irreversible inhibition of the β5 subunit of human 20S proteasome by salinosporamide A (SalA) was studied by means of QM/MM molecular dynamics simulations. The free energy landscape computed at the M06-2X:AM1/AMBER level reveals the existence of a favorable pathway, where Lys33 actively participates in the inhibition process. This mechanism that presents a free energy barrier for the rate-limiting step in agreement with the experimentally measured rate constant is different from the widely accepted SalA-assisted mechanism. Analysis of the electrostatic features of 20S proteasome reveals the importance of the electrostatic preorganization/ reorganization of the enzyme and the pivotal role of Asp17 in modulating pKa of Lys33 and explains how a molecule, completely unlike the natural substrate of 20S proteasome, binds and inhibits its active site. Our results can contribute to the refinement of efficient inhibitors of a key player in the cell protein quality control system, with the consequent potential applications in medical treatments.