Neurolysin (Nln) is a Zn metallopeptidase, known to hydrolyze polypeptides up to 19 amino acids in extracellular conditions. Nln has been shown to modulate the defense mechanism of brain against acute neurodegenerative disorders. For example, over-expression of neurolysin significantly reduces the injury due to brain ischemia. In this context, small molecule activators of Nln have been recently identified which can increase catalytic activity of the peptidase and potentially serve as therapeutic agents. Here, we choose one such activator (L-Histidine-L-Tyrosine (LHLT) dipeptide) and present a thorough study with the aims of: (a) identifying the probable activator binding sites on Nln, (b) understanding the conformational modulations of Nln upon activator binding and (c) specific and allosteric interactions between activator and Nln that may lead to the enhanced catalytic activity. Nln is simulated in an aqueous solution at 300K temperature, 1 bar pressure with 1:100 molecular ratio of Nln:LHLT for 4.5 µs (five separate runs of 900ns each) and final 3 µs are used for the analysis. We ran identical simulations with the (i) apo Nln and (ii) a reported negative control molecule of the LHLT, which is L-Tyrosine-L-Histidine (LTLH). We found out that the activator, LHLT preferentially binds to the lower end of Nln helical channel, disrupting a GLU-LYS saltbridge and keeps the channel in ‘open’ state consistently with higher probability. The lower end of the channel broadens by an average of 2.5 Å (~25%) in comparison to the apo and negative control bound Nln, facilitating access of peptide substrates to the catalytic site in the LHLT bound state. This is further strengthened by a scanning of H-bonding interactions between LHLT and Nln channel region amino acid sidechains, which clearly indicates the specific GLU of saltbridge as the most susceptible point of contact for LHLT. We also observe conformational modulations in the apo, and activator bound Nln in the channel region as well as in the catalytic site. The correlations between the saltbridge opening in the lower end of the Nln channel and catalytic site structural changes reveal details about allosteric effects in the Nln catalytic activity and provide valuable insight for the design of novel Nln activators.

Activator binding in neurolysin: Understanding the conformational landscape and interactions

ACS Spring 2022

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