Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn.
Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.
From the Laboratory of the Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow 119991, Russia.
Vera A
VA
Korneeva
From the Laboratory of the Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow 119991, Russia, the Department of Physics, Moscow State University, Moscow 119992, Russia.
Mikhail M
MM
Trubetskov
the Research Department, HemaCore LLC, Moscow 125319, Russia.
Alena V
AV
Korshunova
the Laboratory of Computer Modeling of Biomolecular Systems and Nanomaterials, Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow 119334, Russia.
Sofya V
SV
Lushchekina
From the Laboratory of the Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow 119991, Russia.
Vladimir N
VN
Kolyadko
the Laboratory of Molecular Diagnostics and Genetic Engineering, Institute of Agricultural Biotechnology of Russian Academy of Agricultural Sciences, Moscow 127550, Russia.
Olga V
OV
Sergienko
the Laboratory of Molecular Diagnostics and Genetic Engineering, Institute of Agricultural Biotechnology of Russian Academy of Agricultural Sciences, Moscow 127550, Russia, the Laboratory of Biologically Active Nanostructures, Gamaleya Institute of Epidemiology and Microbiology of Russian Federation Ministry of Health and Social Development, Moscow 123098, Russia.
Vladimir G
VG
Lunin
From the Laboratory of the Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow 119991, Russia, the Department of Physics, Moscow State University, Moscow 119992, Russia, the Research Department, HemaCore LLC, Moscow 125319, Russia, the Research Division, Scientific Clinical Centre of Pediatric Hematology, Oncology, and Immunology Named after Dmitry Rogachev of Ministry of Health of Russian Federation, Moscow 117997, Russia, and the Department of Translational and Regenerative Medicine, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, 141700, Russia mapanteleev@yandex.ru.
Mikhail A
MA
Panteleev
From the Laboratory of the Molecular Mechanisms of Hemostasis, Center for Theoretical Problems of Physicochemical Pharmacology of Russian Academy of Sciences, Moscow 119991, Russia, the Department of Physics, Moscow State University, Moscow 119992, Russia, the Research Department, HemaCore LLC, Moscow 125319, Russia, the Research Division, Scientific Clinical Centre of Pediatric Hematology, Oncology, and Immunology Named after Dmitry Rogachev of Ministry of Health of Russian Federation, Moscow 117997, Russia, and the Department of Translational and Regenerative Medicine, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, 141700, Russia.
Fazoil I
FI
Ataullakhanov
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015945:Factor XIa / Q000037:antagonists & inhibitors*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D003313:Zea mays
D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015945:Factor XIa; D014357:Trypsin
Blood Coagulation Factors; Enzyme Kinetics; Enzyme Mechanisms; Protease Inhibitor; Protein Expression; Protein-Protein Interactions; Serine Protease