Congo Red bound to α-1-proteinase inhibitor as a model of supramolecular ligand and protein complex

Abstract

The complex formation and structure of α-1-proteinase inhibitor with supramolecular ligand Congo Red was predicted using molecular mechanics and molecular dynamics simulation. A seven-molecule Congo Red ligand was introduced to the cleft in β-sheet “A” of an α-1-proteinase inhibitor in place of the peptide chain fragment (342-358) which occupies this locus in the cleaved form of the inhibitor. The striking similarity of Congo Red and peptide chain (342-358) insertion effects, observed by comparison of root mean square (r.m.s.)–distance plots as protein stability increased, confirmed the reliability of the constructed complex. The binding predicted theoretically for the one available cleft in the β-sheet, limited to a few Congo Red molecules, was verified experimentally. α-1-proteinase inhibitor was chosen for this study because of the known natural instability of its β-pleated sheet, but the model is believed to represent other Congo Red complexes involving proteins whose accessibility for dye penetration may be triggered by function-derived structural alterations or may be generated in unfolding conditions.

Introduction

The increasing use of Congo Red as a specific, nonstandard ligand for proteins stimulates interest in understanding the mechanism of its binding. For years this dye was used only as a stain for cellulose and amyloid (Burgevin et al., 1994; Hurle et al., 1994; Woodcock et al., 1995). Recently observed biological effects of its binding to proteins make it one of the more interesting ligands (Rybarska et al., 1991; Kaszuba et al., 1993; Roterman et al., 1993; Bhat et al., 1994; Elhaddaoui et al., 1995). It is becoming evident that the liquid-crystalline nature of this dye largely determines its peculiar complexation property (Stopa et al., 1997). Despite the supramolecular nature of Congo Red, it binds to a polymeric matrix as a single ligand. The self-assembled molecules of the ligand are usually fixed in clefts of protein β-pleated sheets which become accessible for penetration after being subjected to local or global constraints.

Well-packed native proteins are basically not accessible for dye binding. Triggered penetration seems necessary. In general, susceptibility for binding arises as a consequence of local or global destabilization of third-order protein structure, usually not clearly defined (Roterman et al., 1994). Some proteins, however, present exceptions, among them amyloid and α-1-proteinase inhibitor. They appear capable of binding the dye without being destabilized. While the accessibility and precise location of binding sites in amyloid proteins is still unknown, in α-1-proteinase inhibitors they are easily predictable (Björk et al., 1993; Carrell et al., 1994; Potempa et al., 1994; Hopkins and Stone, 1995; Carrell and Stein, 1996; Chang et al., 1996). Hence, the α-1-proteinase inhibitor was a convenient object for construction of a protein–Congo Red model of the complex. This paper presents the computer-constructed complex, with experimental confirmation.