Structural Determinants in Hinge-Bend Motions of Neurolysin

Neurolysin (EC 3.4.24.16; NLN) is a zinc (II) metallopeptidase, in the M3 family of enzymes, along with close relatives such as thimet oligopeptidase (TOP). NLN is believed to play a role in diverse physiological processes through cleavage of neuropeptides, including neurotensin and bradykinin. NLN and similar enzymes are thought to operate via a hinge-clasp mechanism whereby a substrate enters the active site of the open, inactive form and conformational changes in the hinge region lead the enzyme to adopt a closed, active form. These conformational changes likely provide a mechanism for limiting the length of peptides processed by NLN and TOP. This research will use site-directed mutagenesis followed by spectroscopic and kinetic assays of the engineered enzymes to examine the importance of residues in the clasp region on the mechanism of substrate selectivity and hydrolysis. To this end we have optimized the expression and purification of the cytosolic isoform of mouse NLN – subcloned into a pET28b(+) vector, overexpressed in E. coli, and purified by Ni-affinity chromatography. We have created homology models of the open and closed conformations of mouse neurolysin; these models suggest that alpha helices 3 and 6, located on either side of the hinge region, play a role in modulating the stability of the closed, active form of the enzyme. Using our homology models, we have analyzed this region and identified several potential stabilizing interactions, including a salt bridge between GLU135 and LYS189, residues highly conserved in NLN and similar enzymes. Analysis of the effect of mutations in the clasp region on enzyme stability and activity are currently underway.

Grant Funding Source: This research was funded by the Robert W. and Beverly J. Summers Scholarship through Saint Mary's College of California's Summer Research Program.