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

Scientific Reports
Mekdes DebelaPeter Goettig

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 compr...Continue Reading

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Citations

Oct 23, 2019·Biological Chemistry·Shiyu ChenMatthew Bogyo
Jul 14, 2020·Frontiers in Cellular Neuroscience·Cinthia MellaPamela Ehrenfeld
Sep 11, 2019·Biochimie·Peter GoettigViktor Magdolen
Dec 21, 2020·Neuropathology and Applied Neurobiology·Arne HerringKathy Keyvani

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Datasets Mentioned

BETA
PXD006884

Methods Mentioned

BETA
PCR
X-ray
glycosylation
proteomic
specificity profiling
size exclusion chromatography

Software Mentioned

APBS
Web
XTRIAGE
CTRUNCATE
MODEL
NQ
PHENIX
PyMOL
SCALA
AIMLESS

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