TY  - JOUR
AU  - Cohen, Itay
AU  - Coban, Matt
AU  - Shahar, Anat
AU  - Sankaran, Banumathi
AU  - Hockla, Alexandra
AU  - Lacham, Shiran
AU  - Caulfield, Thomas R
AU  - Radisky, Evette S
AU  - Papo, Niv
TI  - Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin.
JO  - The journal of biological chemistry
VL  - 294
IS  - 13
SN  - 0021-9258
CY  - Bethesda, Md.
PB  - Soc.
M1  - DKFZ-2025-02511
SP  - 5105 - 5120
PY  - 2019
N1  - #DKFZ-MOST-GR-2495#
AB  - Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.
KW  - Amyloid beta-Protein Precursor: chemistry
KW  - Amyloid beta-Protein Precursor: genetics
KW  - Amyloid beta-Protein Precursor: metabolism
KW  - Animals
KW  - Aprotinin: chemistry
KW  - Aprotinin: genetics
KW  - Aprotinin: metabolism
KW  - Crystallography, X-Ray
KW  - Disulfides: chemistry
KW  - Disulfides: metabolism
KW  - Humans
KW  - Models, Molecular
KW  - Protein Conformation
KW  - Protein Domains
KW  - Protein Engineering
KW  - Proteolysis
KW  - Trypsin: chemistry
KW  - Trypsin: metabolism
KW  - crystal structure (Other)
KW  - disulfide (Other)
KW  - molecular dynamics (Other)
KW  - protease inhibitor (Other)
KW  - protein engineering (Other)
KW  - protein structure (Other)
KW  - proteolysis (Other)
KW  - serine protease (Other)
KW  - APP protein, human (NLM Chemicals)
KW  - Amyloid beta-Protein Precursor (NLM Chemicals)
KW  - Disulfides (NLM Chemicals)
KW  - Aprotinin (NLM Chemicals)
KW  - PRSS3 protein, human (NLM Chemicals)
KW  - Trypsin (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:30700553
C2  - pmc:PMC6442025
DO  - DOI:10.1074/jbc.RA118.007292
UR  - https://inrepo02.dkfz.de/record/306285
ER  -