Involvement of disulfide bonds and histidine 172 in a unique β‐sheet to α‐helix transition of α₁‐acid glycoprotein at the biomembrane interface

Abstract

Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes.

The 1.8-Å Crystal Structure of α1-Acid Glycoprotein (Orosomucoid) Solved by UV RIP Reveals the Broad Drug-Binding Activity of This Human Plasma Lipocalin

“…The crucial role of these disulphide bonds for the correct folding of AGP and for its potential interaction with biomembranes was recently demonstrated. 41 Loops 2 and 3 at the entrance to the cavity are rather short and form hairpin turns, whereas loops 1 and 4 are much longer (Fig. 2).…”

“…Surprisingly, loop 1, which is disordered in many other crystal structures of lipocalins such as human tear lipocalin 42 and complement component 8γ (C8γ), 43 is structurally well defined despite its unusual length of 15 residues (according to a previous definition of conserved structural elements in the lipocalins 38 ). Furthermore, this loop exhibits two full turns of an α-helix (residues [35][36][37][38][39][40][41][42][43]. Notably, one of the glycosylation sites (Asn38) occurs within this α-helix, suggesting that changes in the glycosylation pattern might influence its conformation and, thus, control the ligand-binding activity.…”

“…Recent fluorescence and CD spectroscopy studies reported an increase in the α-helical content (from 13.4% to ca 30%) of AGP upon disulphide reduction and also in the presence of biomembranes. 41 As the AGP crystal structure shows an elevated α-helical content compared with other lipocalins and with the earlier CD measurements, 11 it has to be considered whether the crystal packing could effect a corresponding structural change. If we assume, for example, that structural ordering of the C-terminus happens upon crystallization, a calculation of secondary structure content with omission of the corresponding segments (residues 152-154, 160-162, and 166-172) would result in 18.6% α-helix, which is closer to the value reported for the free native protein in solution.…”

Influence of ligand binding on structure and thermostability of human α1-acid glycoprotein

“…Thus, the disulfide bridge might serve as a switch for ligand release. This proposed model of action is in agreement with the importance of S–S bridges for the formation of α‐helices in AGP as well (Nishi et al ., ). Because this structural change was observed to be a prerequisite for the melting of α‐helices, the disulfide bridge between Cys 72 and Cys 165 seems to be the most probable candidate.…”

“…Thus, only half of the helical content of crystallized AGP was observed for native AGP, where the helices are placed on the periphery of AGP being in direct contact with glycan antennas. Taking into account the controversial reported results with respect to the deglycosylation of AGP that range from zero influence with no significant changes in its tertiary structure (Nishi et al ., ) via decreased ordering (Šebánková et al ., ) as far as disruption of protein structure (Albani, ), we can conclude that our results support strong structural influence of the carbohydrate moiety on the protein moiety.…”