Concanavalin A (PDB ID = 3D4K)
Created by Jacqueline Stevens
Concanavalin A (ConA), a legume lectin isolated from Canavalia ensiformis, is a sugar binding protein (1). Plant lectins such as ConA contain at least one non-catalytic domain that binds reversibly to mono or oligosaccharide ligands (8). The molecular weight and pI of ConA are 25598.47 daltons and 5.27 respectively. ConA exists in an alpha-2 homodimeric or an alpha-4 homotetrameric form depending on the pH of the solution (4). Each subunit has one ligand binding-site capable of binding mannose or glucose, however research has shown that mannose has a higher affinity (2). Consequently, a ConA protein complex can bind multiple carbohydrate ligands at once (4). Carbohydrate ligands bind to an evolutionarily conserved ligand binding-site in ConA (3). The specificity of Concanavalin A's binding activity is important for the protein's interaction with glycoproteins (2). ConA binds to sugar components of membranous proteins, such as N-linked glycans, allowing it to function in leukocyte adhesion, tumor metastasis, and pathogen recognition (4). Additionally, ConA and other plant lectins play an important role in plant defense against pathogens, disease, and harmful environmental conditions (3).
Concanavalin A's binding activity renders it an effective agent for lectin based histochemical staining; this is useful in determining the sugar composition of cell surfaces (1). Con A can also be used in affinity chromatography in order to purify proteins (5). While Concanavalin A is a legume lectin, it may be applied in animal cells; it agglutinates erythrocytes and has a mitogenic role in stimulating T-cells (7). Research suggests that the ability of Concanavalin A to bind to specific glycoproteins could be exploited to help treat hepatoma (5). ConA can bind to mannose moieties of glycoproteins on hepatomic cell membranes (5). The glycoprotien is then internalized, triggering cell death via autophagy (5). Subsequently, ConA, a T-cell mitogen, can stimulate an immune response in the liver (5). Finally, Concanavalin A is the most studied Diocleinae plant lectin and is applied as a model or prototypical legume lectin in order to determine the molecular basis for protein-carbohydrate recognition for other lectins(5,6). Protein-carbohydrate recognition is an important mechanism for inter-cellular communication (7).
The binding sites, ligand recognition mechanisms, and tertiary structures of legume lectins are highly conserved (6). Therefore, the lectins in this family share similar primary and tertiary structural components with Concanavalin A. Protein BLAST results indicated significant primary structure similarities between Concanavalin A and Canavalia maritima lectin (ConM; PBD ID 2ow4). This was indicated by a very low E score of 9e-131 for the comparison. A low E value, close to zero, indicated that the similarity was significant. The primary structure of ConM differs from that of ConA by five point mutations. These differences in the primary structure affect the sugar-binding activities of ConA compared to ConM (6). For example, ConM has a higher affinity for binding maltose and trehalose than ConA (6). A single point mutation is responsible for this difference in binding affinities for carbohydrate ligands; Pro-202 in ConA is replaced by Ser-202 in ConM (6). This point mutation results in a functional difference between ConA and ConM; the Pro-202 residue in ConA promotes approximation of Tyr-12 to the carbohydrate binding-site (6). Consequently, ConM, with a different approximation of Tyr-12, has a higher affinity for disaccharides (6).
Dali server results also suggested that ConM has a similar tertiary structure to ConA (9). The tertiary structure of these two proteins has been described as a jelly roll structure (8). A significant Z score of 42.5 indicated this structural similarity. The RMSD for the comparison between ConA and ConM was 0.9Å, and was also significant. Despite the structural similarities, differences in the structures of ConA and ConM can be used to explain differences in the functions of these two proteins. For example, ConM plays an important role in releasing nitric oxide in endothelial cells; ConA does not share this function (8). ConA and ConM also differ in their mitogenicity (8). These functional differences arise primarily from residue differences near ligand-binding sites (8). While ConA and ConM share similar primary and tertiary structures, differences in structure account for their unique functions.
Concanavalin A (ConA) specifically binds mannose-containing carbohydrate ligands. ConA exists in a dimeric or tetrameric form depending on the pH of the solution and is a homotetramer at physiological pH (2). Each monomer contains 237 residues, one saccharide-binding site, and transition metal and calcium binding sites in close proximity to the sugar-binding pocket (10). The substrate specificity of ConA is determined primarily by unique quaternary structure, the oligomeric association of monomeric subunits (11,12). Each monomer consists of two anti-parallel beta-sheets: a six-stranded back beta-sheet and a curved seven-stranded front beta-sheet (12,13,14). These sheets are connected by a five-stranded top beta-sheet, forming a characteristic jelly roll structure (12,13,14). ConA exemplifies this tertiary structure conserved in legume lectins (12,13). For example, Dali server results indicated significant tertiary structural similarity between ConA and Canavalia maritima (ConM; PDB ID 2ow4), which also exhibits the jelly roll structure (9,15). The oligomerization of ConA into dimeric or tetrameric structures can be described based on the association of the tertiary subunits (14).
ConA is comprised of canonical legume lectin dimers, a subunit distinguished by a twelve-stranded beta-sheet interface between individual monomers formed by the side-to-side interaction of the two six-stranded back beta-sheets (11,14). In the tetrameric form, ConA forms a dimer of dimers via back-to-back association of canonical dimer subunits; the two identical dimers are joined through the central portion of their back beta-sheets (12,14,16). At low pH, van der Waals contacts and hydrogen bonds are less prevalent, and the canonical dimeric conformation predominates (11,12). Consequently, the quaternary structure of ConA is dictated by a pH-dependant dimer-tetramer equilibrium (16). Oligomerization allows for different specificities for dimeric and tetrameric ConA (14). For example, the dimer and tetramer have equal affinity for carbohydrates containing 1, 2, or 5 mannose groups, however only the tetramer recognizes and binds higher order carbohydrates with 7, 8, or 9 mannose groups (14). Additionally, recent studies suggest that two Histidine residues (His-51 and His-127) affect the stability and pH dependence of the ConA tetramer (24). These residues may help drive the association of dimeric ConA into the tetrameric form (24). The quaternary structure determines the binding activity of ConA; the dimeric form can bind two carbohydrates while the tetrameric form can bind four.
Concanavalin A is a primarily beta structure (17). Each monomeric subunit also contains four turns approximately resembling alpha helices: Asn-14 to Gly-18, Asp-80 to Val-84, Thr-150 to Gly-152, and Thr-226 to Leu-230 (18). The residues not included in beta-sheet, or helix, are arranged into regions of random coil; these stuctures make up the secondary structure of ConA (17). The carbohydrate binding-specificity of ConA is based on its unique binding loop, which is stabilized in the active (locked) conformation of ConA by the binding of Ca2+ and Mn2+ (19). The transition metal binding site can also bind Ni, Co, Zn, and Cd, but is selective for Mn (1). The transition metal and calcium binding sites are located 12.5Å and 8.5Å from the center of the sugar binding site (1). Interactions with the divalent metal ions result in the formation of the shallow carbohydrate binding site (1,10). Therefore, the reversible binding of Mn2+ and Ca2+ dictates carbohydrate binding (19). Five amino acid residues are essential for metal ion binding: His-24 and Glu-8 in the Mn2+ binding site, Asn-14 in the Ca2+ binding site, and Asp-10 and Asp-19 in a carboxylate group bridge between the two metal ion binding sites (19). Binding of the metal ions induces the conformation change from unlocked (inactive) to locked (active) and maintains the protein in the locked conformation (20).
Concanavalin A also contains many residues essential to the recognition and binding of carbohydrate ligands. The carbohydrate binding site is comprised of Tyr-12, Pro-13, Asn-14, Thr-15, Asp-16, Leu-99, Asp-208, and Arg-228, a conserved sequence in the legume lectin family (2). Of particular interest is the circular permutation of Concanavalin A, which has been demonstrated in tetravalent ConA (11). Circular permutation in ConA is a form of post-translational processing in which a loop segment is removed by jack bean asparaginyl endopeptidase resulting in a circularly permuted protein in which the amino and carboxy termini are ligated, forming a new loop (11,21). The carbohydrate binding loop is formed by Tyr-12, Asn-14, Leu-99, Tyr-100, Asp-208, and Arg-228 (10). Asn-14, Arg-228, and Asp-208 each participate in hydrogen bonds essential to protein-carbohydrate interactions and interact with bound Ca2+ (11). Asn-14 binds carbohydrates via its amide nitrogen and interacts with Ca2+ via an oxygen atom (19). Arg-228 interacts with carbohydrates via a conserved water molecule and with Ca2+ via a water bridge (11,19). Asp-208 is preceded by a cis-conformation of the Ala-207:Asp-208 peptide bond (11). The conformation change from unlocked to locked accompanying the metallization of ConA is governed by a trans-to-cis isomerization of the Ala-207:Asp-208 peptide bond (19). These residues are situated between the Ca2+ binding site and the carbohydrate specificity-determining loop (19). When the metal binding sites are occupied, the side chain of Thr-11 is pushed up against the Ala-207:Asp-208 peptide bond, creating steric strain that drives the trans-cis isomerization (19). The locked conformation exhibits high affinity for carbohydrate substrates such as trimannosyl Man-1,3[Man-1,6]Man in N-linked glycans (11).
Additionally, van der Waals interactions between Tyr-12 and the saccharide ring of a carbohydrate are important for the approximation of Tyr-12 to the carbohydrate binding site (11,22). Differences in primary structures of ConA and ConM result in different sugar binding affinities; Pro-202 in ConA is replaced by Ser-202 in ConM (22). Pro-202 in ConA promotes approximation of Tyr-12 to the carbohydrate binding site (22). Consequently, ConM, with a different approximation of Tyr12, has higher affinity for disaccharides (22). Carbohydrate binding contributes to ConA's ability to interact with glycoproteins, which is essential to ConA's function in leukocyte adhesion, tumor metastasis, pathogen recognition, and inter-cellular communication (2,3,22). Finally, crystal structures of ConA bound to carbohydrate ligands have illustrated the presence of a conserved water molecule in the sugar-binding pocket (PDB ID 3d4k) that mediates carbohydrate binding, effectively anchoring the mannoside-containing ligand (1). The conserved water molecule bridges the interaction between ConA and the mannoside ligand, mediating binding by forming hydrogen bonds with Asn-14, Asp-16, Arg-228 and a mannosyl residue of the carbohydrate ligand (1). Crystal complexes of ConA to trimmanoside glycans (PDB ID 1cvn) verify the importance of the conserved water molecule (23). The sugar residues bind to the monossacharide binding site and to the extended sugar-binding pocket formed by Tyr-12, Pro-13, Asn-14, Thr-15, and Asp-16 (23). Water anchors the reducing sugar unit to the protein (23). These studies verify the high affinity of ConA for N-linked glycans (23).