Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1.lovin.s

Abstract

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.

A common neoepitope is created when the reactive center of C1-inhibitor is cleaved by plasma kallikrein, activated factor XII fragment, C1 esterase, or neutrophil elastase.

“…The stability of aI -antitrypsin or antithrombin is increased by proteolysis of the exposed loop of these serpins (7,14,15); a parallel observation has been made here with C1-inhibitor.…”

“…This process irreversibly modifies the conformation of the serpin molecule, creating sites responsible for the rapid metabolic clearance of enzyme-serpin complexes and for the regulation of serpin biosynthesis (6-1 1). The following observations support the notion that serpins undergo major conformational changes when they are cleaved at or next to their reactive center: (a) complex formation between thrombin and antithrombin reduces the ,B-sheet structure of antithrombin from 6-8 to 1-2% (12); (b) Met358 residue of al-antitrypsin (residue P1) is found 67 A apart from Ser359 (residue PI) when the inhibitor is cleaved at PI-PV, whereas the distance between C and N in an intact peptide bond is 1.3 A (6,13); (c) proteolytic cleavage of the exposed loops ofa 1-antitrypsin, antithrombin, or Cl-inhibitor strikingly increases the molecular stability of these proteins (7,14,15); and (d) Cl-inhibitor that has reacted with plasma kallikrein expresses an epitope that is undetectable on the native serpin molecule ( 16). In the study reported here, we have examined further the consequences for the conformation of Cl-inhibitor of proteolytic cleavage of its exposed loop.…”

The structural basis for neutrophil inactivation of C1̅ inhibitor

“…When such cleavage occurs both antitrypsin and antithrombin III undergo a marked increase in antigenic heat-stability (Carrell & Owen, 1985). A study of Cl -INH denaturation has demonstrated a single transition for the native inhibitor at 60°C but no major detectable transition for the Cl-INH-Cls complex (Lennick et al, 1985).…”

Effects of Serpin Binding on the Target Proteinase: Global Stabilization, Localized Increased Structural Flexibility, and Conserved Hydrogen Bonding at the Active Site

“…The results of thermal and chemical denaturation experiments indicate that both components are more stable in the proteinase-serpin complex than in separated forms (Atha et al, 1984;Lennick et al, 1985;Mast et al, 1991;Komiyama et al, 1994;Enghild et al, 1994) and that in these experiments the behavior of serpins in the complex is similar to that of the cleaved, RSL-inserted form of the inhibitor [see also Shore et al (1995), Lawrence et al (1995), Plotnick et al (1996), Mast et al (1991), Bjo ¨rk et al (1993, Olson et al (1995), and Debrock and Declerck (1995)]. In contrast to these results from thermal and chemical denaturation, we have recently reported that the D189S mutant of rat trypsin, when complexed with human R 1 -proteinase inhibitor (R 1 -PI), becomes more susceptible to limited proteolysis (Kaslik et al, 1995); this indicates that at least the first cleavage site is more accessible to proteinase in the complex than in uncomplexed trypsin.…”