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Sci Rep. 2018 Jul 16;8(1):10705. doi: 10.1038/s41598-018-29058-6.

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

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Max-Planck-Institut für Biochemie, Proteinase Research Group, 82152, Martinsried, Germany.
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
Klinische Forschergruppe der Frauenklinik, Klinikum rechts der Isar der TU München, 81675, München, Germany.
Division of Structural Biology, Department of Biosciences, University of Salzburg, 5020, Salzburg, Austria.
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94158, USA.
Institute of Molecular Medicine and Cell Research, University of Freiburg, 79104, Freiburg, Germany.
BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104, Freiburg, Germany.
German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany.
Division of Structural Biology, Department of Biosciences, University of Salzburg, 5020, Salzburg, Austria.


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.

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