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@ARTICLE{Cohen:306285,
      author       = {I. Cohen and M. Coban and A. Shahar and B. Sankaran and A.
                      Hockla and S. Lacham and T. R. Caulfield and E. S. Radisky
                      and N. Papo},
      title        = {{D}isulfide engineering of human {K}unitz-type serine
                      protease inhibitors enhances proteolytic stability and
                      target affinity toward mesotrypsin.},
      journal      = {The journal of biological chemistry},
      volume       = {294},
      number       = {13},
      issn         = {0021-9258},
      address      = {Bethesda, Md.},
      publisher    = {Soc.},
      reportid     = {DKFZ-2025-02511},
      pages        = {5105 - 5120},
      year         = {2019},
      note         = {#DKFZ-MOST-GR-2495#},
      abstract     = {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.},
      keywords     = {Amyloid beta-Protein Precursor: chemistry / Amyloid
                      beta-Protein Precursor: genetics / Amyloid beta-Protein
                      Precursor: metabolism / Animals / Aprotinin: chemistry /
                      Aprotinin: genetics / Aprotinin: metabolism /
                      Crystallography, X-Ray / Disulfides: chemistry / Disulfides:
                      metabolism / Humans / Models, Molecular / Protein
                      Conformation / Protein Domains / Protein Engineering /
                      Proteolysis / Trypsin: chemistry / Trypsin: metabolism /
                      crystal structure (Other) / disulfide (Other) / molecular
                      dynamics (Other) / protease inhibitor (Other) / protein
                      engineering (Other) / protein structure (Other) /
                      proteolysis (Other) / serine protease (Other) / APP protein,
                      human (NLM Chemicals) / Amyloid beta-Protein Precursor (NLM
                      Chemicals) / Disulfides (NLM Chemicals) / Aprotinin (NLM
                      Chemicals) / PRSS3 protein, human (NLM Chemicals) / Trypsin
                      (NLM Chemicals)},
      ddc          = {540},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:30700553},
      pmc          = {pmc:PMC6442025},
      doi          = {10.1074/jbc.RA118.007292},
      url          = {https://inrepo02.dkfz.de/record/306285},
}