The Significance of Ovalbumin as a Model for the Serpin Family
created by Elefterios Trikantzopoulos
Ovalbumin (1OVA) from Gallus gallus is a member of the serine proteinase inhibitor (serpin) super-family, and is a protein that comprises 60 to 65% of the total protein in egg white. The function of ovalbumin is undetermined; a potential role of OVA is the transport and storage of metal ions (3), as well as the source of nutrition for the growing embryo (2). The molecular weight of ovalbumin is 171,391.45 Da, and its isoelectric point (pI) is 5.19 (7).
The major significance of ovalbumin does not reside in its biological function as a molecule. Ovalbumin provides a major interest for study because its structure provides a model for the structure of the inhibitory serpins. To understand this, a background to the structure and function of serpins is provided.
The serpin super-family is composed of more than twenty homologous proteins. Serpins are found in animals, plants and viruses (3), and analysis of the serpin family’s genes shows serpins evolved from a common ancestor of birds and mammals 310 million years ago (2). Serpins are proteins with metastable conformations (4) that are accompanied by thermodynamic stabilization upon exertion of their inhibitory activity. Serpins comprise of a reactive center that acts as a pseudosubstrate for the target protease. The reactive center lies within an exposed sequence, which is referred to as the reactive center loop (1). The protease binds to the reactive center at a P1-P′1 peptide bond and cleaves the tight complex at that site. The serpin rapidly converts from the native stressed (S) conformation to a relaxed (R) hyperstable structure (2), and as the inhibitor is now free to move, the reactive center loop slides into the middle of the proximal β-sheet. This insertion and inhibitory mechanism provide a major thermostabilization for the serpin protein.
The structure of the post cleavage form of inhibitory serpins is known (1), but the structure of the intact serpins and their reactive centers is not known. Crystal structures of the inhibitory serpin in its native uncleaved form have not been reported (3) due to the metastable conformation of the inhibitor. OVA has the ability to interact with proteases as substrates (3) at the reactive center sequence like all serpin proteins, but OVA differs from other serpins because it is non-inhibitory and lacks a loop insertion mechanism. Moreover, ovalbumin lacks the conformational change on cleavage at the P1-P′1 site, and so it provides stability that inhibitory serpins lack, that makes OVA an excellent choice as a model of the intact structure for the serpin super-family (1).
Ovalbumin exists physiologically as a tetramer (3) with four subunits of identical primary structure (5). Each subunit is composed of 386 amino acid residues. OVA does not have a standard N-terminal leader sequence, but the four subunits of OVA share a hydrophobic sequence between residues 50 and 66 that acts as an internal signal sequence involved in transmembrane location (3). Subunit D of OVA contains the reactive center of the protein at the residues Ala-358/Ser-359; the reactive center is found on the final turn of a three-turn α-helix (helix R) formed by Ser-350 (phosphorylated) to Ser-359 (3). The sequence of ovalbumin’s reactive center that forms the peptide stalk at the N-terminal side of helix R (residues 344 to 350) differs from the sequence of the inhibitory serpins at the equivalent position. The reactive center of inhibitory serpins is usually composed of small hydrophobic residues such as Ala and Gly, which suggests that the segment is flexible and capable of undergoing the described loop mechanism. The reactive center of ovalbumin is composed of a large polar Arg at the residue 345, as well as Val at residues 347 and 348, which suggests that the segment is restricted in its movement (3). This difference in the structure of OVA with inhibitory serpins correlates to the function and lack of inhibitory activity of OVA.
Although ovalbumin is a non-inhibitory serpin that lacks a loop insertion mechanism, the ovalbumin mutant R339T contains the loop insertion ability while still retaining the non-inhibitory activity of ovalbumin. The mutant of OVA has the hinge residue Arg-339 of OVA replaced by the less bulky residue of threonine. This change in primary structure transforms the ovalbumin mutant into a considerably thermostabilized form following the P1-P′1 cleavage. The thermostabilized ovalbumin mutant assumes the fully loop-inserted conformation, which implies that ovalbumin also has the serpin metastable nature in the native form (4).
The two ligand components for ovalbumin are calcium ion (Ca2+) and N-acetyl-D-glucosamine (NAG) (5). The functions of calcium ion and NAG are not referred in any sources, but they can be rationally determined. Ca2+ induces crystallization and provides clues to the function of OVA for the transport and storage of metal ions (1). A molecule of NAG is covalently linked to the nitrogen amide of Asn-298 (3) of each subunit, so it can be deduced that NAG functions for the folding of OVA by n-linked glycosylation of the described residues.
The four subunits of OVA do not share identical secondary structures. Subunit A is 32% alpha-helices, 32% beta sheets and 36% random coils. Subunit B is 30% alpha-helices, 32% beta sheets and 38% random coils. Subunit C is 28% alpha helices, 31% beta sheets and 41% random coils. Subunit D is 31% alpha-helices, 31% beta sheets and 38% random coils (5). Ovalbumin has main-chain hydrogen bonds between residues 345 and 346 (the base of N-terminal stalk of helix R), residues 194 to 197 (the top of strand s3A) and residues His-337 O-N Phe-190 (3). Ovalbumin shares a tertiary structure with the serpin super-family proteins consisting of nine α-helices, three beta sheets (2), and the α-helical form of the exposed and mobile reactive center loop that serves as bait for target proteases (1).
An important alternate conformation of ovalbumin is S-ovalbumin (1UHG). A proportion of ovalbumin in stored eggs undergoes conformational change to S-ovalbumin, which is accounted for by an elevation of egg white pH due to the release of carbon dioxide through the eggshell. This has led to the structural mechanism for the s-ovalbumin formation as a puzzling question in food science and serpin structural biology. The structure of S-ovalbumin is almost identical to that of wild type ovalbumin. The lack of the loop insertion ability of ovalbumin remains because the native structural characteristic of Arg-339 is retained. The structural differences of S-ovalbumin are the motion of the preceding loop of strand 1A away from strand 2A, and the changes in the side conformation of Phe-99 and Met-241. The conformational change in the Phe-99 side chain induces a decrease in the solvent accessibility of the surrounding residues. The residues Ser-163, Ser-234 and Ser-319 of ovalbumin take the D-amino acid residue configuration in S-ovalbumin. These chemical inversions are related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin (4).
Alpha-1-antitrypsin (2QUG) of Homo sapiens is a member of the serpin family that has a 30% sequence identity with ovalbumin (3). In contrast to ovalbumin, antitrypsin is an inhibitory protein that, in interaction with a protease, undergoes a tertiary structure conformation on reactive center cleavage. The difference in the capability of antitrypsin’s inhibitory activity is due to the different structure of antitrypsin’s reactive center loop. The specificity of inhibition for serpins is defined by the P1 site (1), and antitrypsin, which primarily inhibits elastase, has the smaller Met residue at the P1 of the reactive center loop (residue 358) instead of the bulkier Arg residue that ovalbumin has. This difference in the structure of antitrypsin about its reactive center loop allows antitrypsin to undergo a conformational change and function as an inhibitory protein.
The Dali server generated a comparison protein based on a protein query with similar tertiary structure, and the results of DALI (Z= 44.8) show that antitrypsin has tertiary similarities to ovalbumin (8). The Dali server uses a sum-of-pairs method, which produces a measure of similarity by comparing intramolecular distances. Similarity is measured by Dali-Z scores; a Z-score greater than two means a protein has similar tertiary structure. PSI-BLAST does not include antitrypsin as a subject in the search query for ovalbumin (9), which means that antitrypsin does not have primary similarities to ovalbumin. PSI-BLAST runs a protein query for proteins of similar primary structure. An E-value is assigned based on gaps of amino acids that the query does not share with subject. The E-value decreases with total sequence homology, and increases with the presence of gaps. An E-value of less than 0.05 indicates significant similarity in primary structure. Subjects are chosen for comparison to protein query that have a PDB structure associated to them, which is denoted by the symbol “S” in the link column of the PSI-BLAST search. ExPASy is a bioinformatics resource portal that provides access to scientific databases and software tools, and one of these tools is the computation of theoretical isoelectric point and molecular weight for a list of UniProt Knowledgebase entries or for user entered sequences.
Antitrypsin is a good example of an inhibitory member of the serpin family, and so a good example of the significance of ovalbumin as a model for the structure of the serpin super-family. The serpins include proteins involved in the regulation of inflammation, apoptosis, angiogenesis, and embyogenesis. Thirteen of these proteins are found in Homo sapiens (2), of which alpha-1-antitrypsin is a member. Antitrypsin is involved in the prevention of alveolar damage in the lungs of the human species, and so the understanding of the structure of antitrypsin, which is contributed by ovalbumin as a model for the serpin super-family, is beneficial to the understanding of the described inhibitory mechanism of antitrypsin. The target protease of antitrypsin is elastase, a protein-cleaving enzyme essential to tissue repair that can also attack and break down the elastin of the alveolar walls if it spreads from the site of inflammation repair, which is usually a consequence of alpha-1-antitrypsin deficiency and emphysema.
The Met-358 residue of antitrypsin functions as a bait reactive center for the elastase, and elastase binds to the loop at the Met residue and cuts the peptide loop. The loop is free to move and slides into the middle of a large β-sheet; this causes the elastase to drag to the opposite side of the antitrypsin structure. At this new binding site, the elastase structure is distorted, and it cannot complete its reaction (6). This inhibitory mechanism is one that serpins share, but one that ovalbumin lacks due to its incapability of undergoing a conformation change. But the understanding and mapping of the structure of the non-inhibitory ovalbumin provides a model to the structure of inhibitory serpins such as antitrypsin prior to their structural conformation, which has led to a better understanding of the function of the serpin super-family as inhibitors.