Crystal structures of neurolysin, which is a zinc metallopeptidase (neuropeptidase), do not show significant conformational changes upon the binding of an allosteric inhibitor. Neurolysin has a prolate ellipsoid shape with a deep channel that runs almost the entire length of the molecule. In this deep channel, neurolysin hydrolyzes the short neuropeptide neurotensin to create inactive shorter fragments and thus controls the neurotensin level in the tissue. The protein is of interest as a therapeutic target since changes in neurotensin level have been implicated in cardiovascular and neurological disorders and cancer, and inhibitors of neurolysin activity have been developed. Therefore, understanding the dynamical and structural differences between the apo and inhibitor-bound forms of neurolysin can aid in further design of potent inhibitors and activators. For this purpose, we performed several molecular dynamics (MD) simulations for both apo and neurolysin bound to an allosteric inhibitor. The collective dynamics of neurolysin is examined using Principal Component Analysis and Elastic Network Model calculations, and common collective motions are revealed in both neurolysin forms. MD simulations show that allosteric inhibitor binding induces additional low-frequency motions that are not observed in the apo form. ENM calculations reveal changes in inter- and intra-domain communication upon binding. Furthermore, differences are observed in the inhibitor bound neurolysin contact network that are far from the active site, which reveals long range allosteric behavior in neurolysin. Finally, a machine learning method Linear Discriminant Analysis is applied to reveal differences between the apo and inhibitor bound ensembles in an automated way, and large differences are observed on residues that are far from both the active site and the inhibitor binding site, which are also in good agreement with the contact network results. This study, using different computational methods on neurolysin, can provide insight into the allosteric modulation of other neuropeptidases with similar folds.

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