Crystal Structure of S-ovalbumin as a Non-loop-inserted Thermostabilized Serpin Form

Abstract

Ovalbumin, a non-inhibitory member of serine proteinase inhibitors (serpin), is transformed into a heatstabilized form, S-ovalbumin, under elevated pH conditions. The structural mechanism for the S-ovalbumin formation has long been a puzzling question in food science and serpin structural biology. On the basis of the commonly observed serpin thermostabilization by insertion of the reactive center loop into the proximal ␤-sheet, the most widely accepted hypothetical model has included partial loop insertion. Here we demonstrate, for the first time, the crystal structure of S-ovalbumin at 1.9-Å resolution. This structure unequivocally excludes the partial loop insertion mechanism; the overall structure, including the reactive center loop structure, is almost the same as that of native ovalbumin, except for the significant motion of the preceding loop of strand 1A away from strand 2A. The most striking finding is that Ser-164, Ser-236, and Ser-320 take the D-amino acid residue configuration. These chemical inversions can be directly related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin. As conformational changes of the side chains, significant alternations are found in the values of the 1 of Phe-99 and the 3 of Met-241. The former conformational change leads to the decreased solvent accessibility of the hydrophobic core around Phe-99, which includes Phe-180 and Phe-378, the highly conserved residues in serpin. This may give a thermodynamic advantage to the structural stability of S-ovalbumin.

The ovalbumin serpins revisited: Perspective from the chicken genome of clade B serpin evolution in vertebrates

“…During embryogenesis, ovalbumin changes to a more heat-stable conformation, while migrating from egg white to amniotic fluid, embryonic organs, and yolk (28). Whether this conformational change of ovalbumin is related (29) or unrelated (30) to classical serpin rearrangement is disputed. Relative to ovalbumin, the paralogues gene Y and gene X have hinge regions that deviate less from the consensus, and these genes, particularly gene Y, may encode inhibitory serpins.…”

Establishment of a Two-Dimensional HPLC-MS/MS Method Combined with DCl/D₂O Hydrolysis for the Determination of Trace Amounts of D-Amino Acid Residues in Proteins

“…Concerning OVA, S-OVA, the thermal stabilizing form of OVA, is produced during the storage of eggs. Yamasaki et al reported that Ser-164, Ser-236, Ser-320 were changed to the D-form in the S-OVA [14]. The estimated %D is 7.9%.…”

“…Because Asp residues can be easily transformed into succinimidyl intermediates, the chiral inversion from L-Asp to D-Asp frequently occurs, therefore, D-Asp residues are often observed in disease/aged proteins [6]. However, some other D-amino acid residues, such as D-Ser and D-Asn, were also formed in a few protein samples [13][14][15]. These reports suggest that various D-amino acid residues are likely to occur without the formation of succinimidyl intermediates.…”

Protein‐Engineering Study of Contribution of Conceivable D‐Serine Residues to the Thermostabilization of Ovalbumin under Alkaline Conditions

“…For the serine residue, rate constant for inversion between l-and d-configuration was reported to be almost the highest among the other residues except cysteine [14]. In the case of S-ovalbumin, only three serine residues in 385-residues polypeptide are determined to be in d-configuration by X-ray crystallographic analysis [10]. In the present study, the effect of point mutation at the conceivable d-serine residues (S164, S236, and S320) was investigated to estimate their contributions to thermostability of S-ovalbumin.…”

“…During the last decade, some observations were additionally reported [6 -12], but still conclusive explanation has not been achieved so far. Among them, our X-ray crystallographic study [10] did offer some possibilities from unique stand points. S-…”