Vicilin
(PDB ID: 4LEJ) from Pinus koraiensis
Created by:
Ethan Arrington
Vicilin (PDB ID: 4LEJ) from Pinus koraiensis is a 7S seed storage unit, which unlike
other similar proteins possesses a novel copper ligand (1). Belonging to
one of the most diverse families of proteins, the cupin-family, Vicilin has a
myriad of possible enzymatic functions (3). Sequence analysis of Vicilin
showed possession of residues coding for IgE epitopes similar to those present
in potent allergens such as the Ara h1 protein, found in peanuts (1,6). The
number of known allergens added by the FDA is expanding every year, however,
little is known about the mechanism of these allergens. Currently, both
the mechanism and exact nature of Vicilin’s allergen-causing role call for
further investigation. Recent structural advancements will hopefully serve to
elucidate Vicilin’s mysterious function and mechanism as a potential allergen.
Vicilin was crystallized to a resolution of 2.7Angtsroms through the hanging-drop vapor-diffusion technique. Crystallization was achieved in a 2M sodium formate, 0.1 M sodium acetate trihydrate solution at pH 4.6 after waiting a period of a week (1,2). X-Ray diffraction was used for structural analysis, following which a structural model of the protein was developed. 95% of all residues were located within the structure. Of the 355 amino acid residues located within the electron density map, only the location of eight residues at the N-terminus and nine residues at the C-terminus remain unable to be located in the structure. Furthermore, The residues (Arg-241, Asp-261, Glu-342, His-355) form two flexible loop regions whose locations within Vicilin remain unknown (1). While unable to be mapped in the structure, the flexible loop regions could be either involved in the existing coordination of a ligand or perhaps serve to coordinate an additional ligand.
The structure of Vicilin is the crux of its function. As shown through Expasy software, the theoretical molecular weight is 46kD and isoelectric point is 5.4 (10). Vicilin only contains one alpha subunit, one glycerol and one phosphate ion. A unique and crucial structural feature is the incorporation of copper ligand. Determining the crystal structure of Vicilin revealed key similarities between it and other proteins in the 7S-storage seed domain, and also revealed several key differences. The N-terminus and the C-terminus form cupin folds, a unique structural motif to this class of protein where a conserved beta barrel is formed (3). Specifically, the intrinsic nature of 7S storage proteins to assume cupin folds remains the foremost area of interest regarding their allergenicity. Cupin folds cluster in seed storage proteins, and possession of a metal ligand imparts a catalytic functionality on the protein. The Cupin fold that is assumed by both the C-terminus and the N-terminus are symmetrically centered off of a psuedodyad axis (1). The axis formation for Vicilin is important in the head to tail arrangement seen in tertiary structure formation, which ultimately allows for the coordination of its novel copper ligand.
Vicilin contains
one alpha subunit. The alpha
subunit itself does not possess any inherent biological function. A trimeric
structure of Vicilin consisting of repeating alpha subunits constitutes the
known biologically active form (Figure 2). The alpha subunits are linked in a
head to tail manner and all feature coordination of a copper ligand. The secondary structure of
Vicilin possesses four major structural motifs. These four major structural
motifs include alpha helices, beta sheets, 3/10 helices and random coils (1). The
four structural motifs constitute the monomeric structure of Vicilin. The
function of Vicilin is most likely closely related to the assumption of a cupin
fold. The conserved beta barrel structural motif of cupin proteins, like
Vicilin, is formed through beta strands (3). Furthermore, Vicilin’s formation
of a conserved beta barrel through beta-strands is also an important aspect of
its ability to coordinate a copper ligand (1,4). The secondary structure of
Vicilin is crucial in that it allows for the protein to bind to copper and more
importantly, not being overly complex allows for it to easily form the
biologically active complex.
The novel finding
of a copper ligand is
paramount to gaining an improved understanding of Vicilin’s function. Vicilin
has several functionally important residues, four of which are involved in the
coordination of the copper ion. As a member of the cupin-family of proteins,
the coordination of a metal ligand is common, however, Vicilin is currently the
only known 7S seed storage protein to possess copper as opposed to other
ligands (1). The coordination of copper has been an important focus as cupin
proteins carry out specific enzymatic functions in plants. These specific
enzymatic functions, which are coordinated through Copper (CuII+), could help
to elucidate the exact function and mechanism of Vicilin.
The copper center of Vicilin contains four primary residues, which include Cys-338, Tyr-67 and two histidine residues, His-340 379, respectively (Figure 1). The coordination of the copper ligand lies in a trigonal planar structure formed though the cysteine’s sulfur, the His-340's ND1 and NE2 of His-379 (1). The hydroxyl group of Tyr-67 forms a fourth bond with the copper and possess a longer coordinating distance than the other three bonds, which is typical of Cu II+ like those found in Vicilin (4). Coordination of copper through histidine residues is linked with enzymatic activity. Typically copper ligands function in electron transfer and oxygen transport and once again impart some kind of catalytic function to a protein (4). Copper’s specific function and potential catalytic role in Vicilin is still under investigation.
The copper center of Vicilin possesses unique characteristics. Type I copper centers typically have strong absorbance at 600nm and function in electron transfer. The type I centers are coordinated by two histidines, and one cysteine residue. Type II centers lack strong absorbance at 600nm and are coordinated by only four cysteine residues (4). As previously detailed, the copper center of Vicilin contains characteristics of both(1).
Vicilin lacked
the strong absorbance of blue light at 600nm, while still possessing a cysteine
residue. Both the possession of a copper ligand and furthermore one that has
unique properties prove intriguing in regards to the function that is specific
to Vicilin. In addition to a potential role in allergenicity, the copper
coordination of Vicilin could potentially be a factor in structural stability.
The copper center’s ability to bind to both N-terminal residues and C-terminal
residues provides further validation into the potential role of copper in its
function.
Adzuki Bean 7S
Globulin-3 (PDB: 2EAA)
is a protein found in Vigna angularis, which is similar to Vicilin and
is a member of the cupin family. Similar primary structure between Globulin-3
and Vicilin was confirmed through the PSI-Blast Software, which is designed
specifically for comparing proteins. The primary structure of nucleic acids can
be compared through using n-Blast. PSI-Blast provided an e-score of 9e-162 for
similarity between the primary structures of Vicilin and Globulin-3 (8). For
PSI-Blast software, the smaller the Z-Score the higher the degree of homology. Similarity between
secondary structure was determined through the Dali Server. The Dali Server
compares the protein of entry among all the proteins registered in the Protein
Data Base. Globulin 3 found in the Adzuki Bean expressed a Z-Score of 42.4
(9). A Z-score greater than two signals a high degree of homology between proteins.
The Adzuki Bean
protein Globulin-3 consists only of a monomeric alpha subunit
that that also possesses a cupin-fold that forms a highly conserved beta
barrel. The monomeric subunit possesses the same symmetry as Vicilin and is
centered off a characteristic pseudo-dyad axis (5). In addition to the cupin
fold Globulin-3 possesses the same secondary structural motifs
including alpha helices, beta barrels, 4/10 helices, and random coils.
Globulin-3 is also thought to have an important role as a potential allergen in
peas, and nuts (5). Lastly, the Adzuki Bean 7S Globulin-3 protein displays a
great deal of homology in the tertiary structure. While the Globulin-3 of the
Adzuki bean possesses many similarities in structure, it does not possess the
novel copper ligand that Vicilin does, however, does possess a metal ion. Like the
catalytic role of copper in Vicilin, calcium plays a catalytic role in the
function of Globulin-3. Furthermore, Globulin-3 also possesses three ligands, Citric Acid, Acetic Acid and Calcium (5).
Vicilin’s role as
a potential allergen in Pinus Korainesis remains grounds for further
investigation. The alignment of Vicilin’s sequence with allergens such as
Ara-h1, signaling expression of IgE epitopes, does indeed show possession of
inherent allergenic characteristics. Vicilin’s possession of a copper ligand is
not only unique in that it is the only known 7S storage protein to possess one,
but also in that it is a novel type of copper center. Further investigation of
Vicilin will hopefully elucidate the root and mechanism of a growing list of
allergens.