TY - JOUR
T1 - Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin
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
N1 - Funding Information:
Acknowledgments—The structural studies of the mesotrypsin-S195A/APPIM17C/I18F/F34C complex were performed on beamline ID30-B at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The structural studies of the mesotrypsin-S195A/ KD1TFPI1K15R/I17C/I34C complex were performed on beamline 5.0.1 of the Berkeley Center for Structural Biology at the Advanced Light Source, Lawrence Berkeley National Laboratory. We are grateful to the beamline scientists for providing assistance in using these beam-lines. X-ray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source. The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231. The Pilatus detector on 5.0.1 was funded under National Institutes of Health Grant S10OD021832. The ALS-ENABLE beam-lines are supported in part by the National Institutes of Health, NIGMS Grant P30 GM124169. X-ray diffraction data were also measured at beamline ID30-B at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.
Funding Information:
This work was supported by the European Research Council “Ideas Program” ERC-2013-StG Grant 336041 (to N. P.), a Prostate Cancer Foundation grant (to N. P.), an Israel Science Foundation (ISF) grant (to N. P.), DKFZ-MOST Grant GR2495 (to N. P.), National Institutes of Health Grant R01CA154387 (to E. S. R.), and a United States–Israel Binational Science Foundation grant (to N. P. and E. S. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 American Society for Biochemistry and Molecular Biology Inc. All Rights Reserved.
PY - 2019/3/29
Y1 - 2019/3/29
N2 - 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.
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.
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U2 - 10.1074/jbc.RA118.007292
DO - 10.1074/jbc.RA118.007292
M3 - Article
C2 - 30700553
AN - SCOPUS:85063957178
VL - 294
SP - 5105
EP - 5120
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 13
ER -