Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin

Mekdes Debela,Viktor Magdolen,Wolfram Bode,Oliver Schilling,Hans Brandstetter,Peter Goettig,Martin L Biniossek,Charles S Craik,Wolfgang Skala,Brigitta Elsässer,Eric L Schneider

doi:10.1038/s41598-018-29058-6

Abstract

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Highlights

Kallikrein-related peptidase 8 was named neuropsin, since the mouse ortholog Klk[8] or mK8 had been cloned from mouse brain, in particular as hippocampus cDNA1
According to SDS-PAGE analysis, this recombinant KLK8 was more than 95% pure, and its integrity was shown by N-terminal sequencing and mass spectrometry
Since only scattered information on natural or synthetic KLK8 substrates was available, the extended substrate specificity of KLK8 was determined by a positional-scanning screening of a synthetic diverse tetrapeptide library (PSSCL) and the proteomic identification of cleavage sites (PICS), which employs a peptide library from digested E. coli proteins[26,27]

Summary

Introduction

Kallikrein-related peptidase 8 was named neuropsin, since the mouse ortholog Klk[8] or mK8 had been cloned from mouse brain, in particular as hippocampus cDNA1. Further studies suggested that mouse Klk[8] (mKlk8) participates in important brain processes, such as neural plasticity and memory formation, whereby long term potentiation (LTP) and the formation of synaptic boutons in mice require the active mKlk[8] protease, which remodels the extracellular matrix[11]. During LTP the activity of voltage-gated calcium channels correlates with elevated extracellular Ca2+ concentration, which has a stimulatory effect on the enzymatic activity of KLK87,13. At synapses it cleaves the cell adhesion molecule L1CAM, neuregulin-1, a signal ligand of the ErbB4 receptor, and the tyrosine kinase EphB2, which modulates the expression of the NMDA receptor[12]. In order to explore human KLK8, the recombinant enzyme from an E. coli expression system was used for X-ray and enzymatic studies, which are the basis for a comparison with the structure of mKlk[824] and for a mechanistic analysis of the structure-functional modules of this intriguing protease

Methods

Results

Conclusion