apo_aequorin

Calcium-loaded apo-aequorin (PDB ID: 1SL8) is isolated from the jellyfish Aequorea victoria, in the luminous coelenterate marine phylum. It exists physiologically as a dimer (1). Aequorin is a Ca²⁺-regulated photoprotien that produces a blue bioluminescence emission in the presence of trace amounts of Ca²⁺ (10). Most Ca²⁺-regulated photoproteins are comprised of a single polypeptide chain of about 22 kDa (7). This is consistent according to ExPASy, a bioinformatics resource portal to many other scientific databases and tools, indicating the molecular weight of the protein to be 2186.15 Da and its isoelectric point to be 4.90 (3). Its bioluminescent characteristic is used for many biological and medical uses such as imaging and targeting since it can be modified to include specific targeting sequences (12). Primarily, it is used as an indicator for studying intracellular calcium levels and distribution in real-time (6). For the jellyfish, the bioluminescence is said to have some survival benefit but the actual nature of the benefit is still unknown (9).

The secondary structure of the aequorin is comprised α-helices, β-sheets, and random coils. It is comprised of one subunit, A, containing three canonical Ca²⁺-binding EF-hands (1). As shown in Figure 1A, it has a globular tertiary structure that contains four helix-loop-helix motifs: I and II are in the N-terminal whereas III and IV are in the C-terminal region (7).The unique structural feature of EF-hand proteins in Ca²⁺-binding are the helix-loop-helix motif where two helices surround a conical loop made up of 12 continuous residues (1). The EF-hand motif usually occurs in pairs, which is significant for structural folding and increased Ca²⁺ affinity (11). The paired EF-hands exhibit extensive hydrophobic interactions between the two pairs and a short β-interaction between the two binding loops. The EF-hand motif I for photoproteins is paired with motif II, which does not have the conical sequence for Ca²⁺-binding even though it has the characteristic structural features of the motif (Figure 1A).

The EF-hand conical region allows for the oxygen ligands in the calcium ion pentagonal bipyramidal array coordination (1). Once the Ca²⁺ binds to the EF-hand structures, an intramolecular oxidative decarboxylation reaction occurs in which coelenterazine, an imidazopyrazine derivative compound non-covalently bound within the protein, is oxidized to coelenteramide (1, 5). Along with the coelenteramide, it produces a blue light emission, a blue fluorescent protein (BFP), and CO₂ (6). There are five different known conformation states depending on the binding of various ligands (1). The five states are shown in Figure 1B: state I is the apo-protein; state II is the photoprotein with the coelenterazine, which is the active state created by incubating the photoprotein with coelenterazine in the presence of O₂; state III is the product of the bioluminescence reaction, BFP, with coelenteramide and bound Ca²⁺; state IV is the photoprotein without Ca²⁺ but with coelenteramide; and state V is the Ca²⁺-loaded apo-protein conformation (1). When conforming to state V, the EF-hand loops shift their positions and the Ca²⁺ is found in the center of the oxygen molecules forming a pentagonal bipyramid geometrical arrangement (1). The binding sites contain six oxygen ligands from peptide backbone carbonyl groups, carboxylic side chains of Asp and Glu residues, a hydroxyl group of Ser, or the side chain of Asn while the seventh oxygen ligand comes from a water molecule (1). One or no water ligands in the EF-hands indicates high affinity Ca²⁺-binding sites (1). These residues are all polar and creates a hydrophillic binding site where the Ca²⁺ligand can hydrogen bond.

His-175 (171 in Aequorea victoria) is found in α-helix of the EF-hand loop IV, and it is involved in a critical step for bioluminescence initiation (9). The trigger occurs from the spatial displacement of this residue, when the significant conformational change occurs in loop IV when accommodating for Ca²⁺. Ca²⁺-binding into the loops of the EF-hands propagates into the active site of the protein when occupied by the coelenteramide (1). His-171 resides towards the end of helix H before the C-terminus of the protein (Figure 1A). In state II, an Arg hydrogen bond binds together helix A and the C-terminus, which caps the substrate cavity containing the coelenterazine, resulting in a solvent-inaccessible and nonpolar environment (9). Similarly, His–Trp and Arg–Phe hydrogen bonds bind together helix A and helix H (1). In state V, the hydrogen-bond network is completely different compared to state II, and there is a significant repositioning and flipping of the His-171 imidazole ring (9). This configuration change decreases the distance between His-171 and Tyr-190 (186 in Aequorea victoria) and allows for a partial positive charge on His-171 and a negatively charged Tyr-176 phenolate form (9). With the hydrogen bonding between His-22 (20 in Aequorea victoria) and the coelenterazine preserved after Ca²⁺-binding, rapid excited-state proton transfer can occur, leading to the excited state of the phenolate ion pair that is responsible for the blue emission (9).

A query was run in two algorithmic protein databases, the Dali server and the Position-Specific Integrated Basic Local Assignment Search Tool (PSI-BLAST), to find a comparison protein for calcium-loaded apo-aequorin, called calcium-loaded apo-obelin (PDB ID: 1SL7) – from the hydroid Obelia longissima. The Dali server runs a query based on tertiary structure similarity via a sum-of-pairs method to calculate intermolecular distances. The output is a Z-score which indicates significant similarity if greater than 2. The Z-score given by the Dali server for the comparison protein was 19.9, indicating high tertiary similarity between the two (2). The PSI-BLAST runs a query based on the primary structure similarity by looking at the gaps in the amino acid sequence of the subject protein in comparison to that of the query protein. The generated output is an E value which is indicative of high similarity if the value falls below 0.5. The E-value generated by PSI-BLAST was 4e-91 for the subject (195 amino acids) in comparison to calcium-loaded apo-aequorin (191 amino acids) (3). This also indicates high similarity between the two proteins, specifically in the primary structure.

Both photoproteins are members of the EF-calcium-binding protein family. Even though this grouping occurs on the basis of structural similarity instead of function, both are bioluminescent proteins that produce a blue light (1). Along with functional similarity, there is substantial structural homology (66%) between the two proteins. A comparative amino acid sequence analysis revealed that one of the ORF’s from apo-obelin (ORFI) is similar to that of apo-aequorin and that Cys-158 and Cys-161 are conserved between these two photoproteins (5). In the bioluminescence spectra for the Ca²⁺-regulated photoproteins, apo-aequorin has a λmax = 469 nm and apo-obelin has a λmax = 482 nm (8). This wavelength distinction is due to apo-aequorin having a hydrogen bond formation between Tyr-82 (84 in Aequorea victoria) and the bound coelenteramide, which is not present in apo-obelin at the corresponding Phe-88 (8).

Though both photoproteins are in the same conformational state V and share a very similar overall fold, local structural differences are found in almost every part of the helical and loop structures (1). The root mean square deviation (RMSD) of C_α of the two apo-proteins in conformation state V is 2.27 Å while it is 1.48 Å between the two photoproteins (state I) from residues 15-181(1). Loops I and II from the EF-hand motifs have similar interactions in both photoproteins in state II, but once Ca²⁺-binding occurs in state V, differences in hydrogen bonding give rise to the observed difference in bioluminescence kinetics between the two photoproteins (10). In both photoproteins, they are bound via hydrogen bonds between the backbone nitrogen and carbonyl oxygen of Ile-37 and Ile-83, the backbone nitrogen of Gly-80, and between side-chain nitrogen of Lys-36 and the side-chain oxygen of Glu-82 (1). However, in state V, the hydrogen-bond pattern of the loop–loop interaction changes when Ca²⁺-binding in loop I breaks the Lys-36 and Glu-82 hydrogen bond (Lys-41 and Glu-83 in Aequorea victoria) (1). In apo-obelin the hydrogen bond distances between the backbone nitrogen of Ile-37 and the backbone carbonyl oxygen of Ile-83, and vice versa of the same residues are 3.03 Å and 2.80 Å, respectively; in state V, the distances change to 2.91 Å and 3.03 Å. Yet, for the Ca²⁺-loaded apo-aequorin, both distances decrease (1). The hydrogen bond distances between Asp-40 oxygen and the backbone nitrogen of Gly-80 that bind two helices in apo-obelin remain the same, whereas the distance increases in apo-aequorin with Ca²⁺-binding (1). Similar changes in interaction occur between loops III and IV. This indicates that the similarity between the photoproteins is greater than the Ca²⁺-loaded apo-proteins.

The calcium-free state of obelin is more pre-positioned than aequorin and as a consequence, obelin is faster in responding to Ca²⁺ concentration changes than aequorin (10). Loop I has the highest affinity for Ca²⁺ of the loops in obelin since they are better positioned for Ca²⁺-binding, requiring minimal shifts to accommodate the ligand. Loop I does not hold the usual pentagonal bipyramidal configuration as the invariant ligand Glu-41 hardly shifted from its position in obelin, causing greater affinity in comparison to loops III and IV (1). In loop III, though the residues are closely oriented, further adjustment is required for proper Ca²⁺-binding since the RMSD between state V and state II of the residues in this loop is higher than that of loop I (1). The greatest displacement occurs in Glu-12 (1). Of the three loops, loop IV experiences the greatest residue reorientation and repositioning which yields the lower affinity Ca²⁺-binding (1). Though the homology is maintained, the loop regions of the EF-hand motifs I and III of aequorin in state II are not as geometrically optimized as the obelin protein for accommodating Ca²⁺-binding in state V; therefore, several residue side-chain ligands must be reoriented instead of the bidentate glutamate ligand, which has to move significantly in apo-obelin (1). More conformational changes are required for Ca²⁺-binding in loop region IV in aequorin than in obelin. This difference is consistent among all the loop regions.

Furthermore, in obelin the hydrogen bonds binding together helices A and H are gone in state V and there are only two hydrogen bonds remaining between Arg-21 and Arg-17 and the residues from the C-terminus (1). The different character of changes in hydrogen-bond interactions between these two α-helices and between helix A and the C terminus in aequorin and obelin further support the different conformational transients on Ca²⁺-binding. Overall, the binding of Ca²⁺ is not independent to the other loops and they are affected differently based on residues present. There are also interactions between helices on different loops, which are also dependent on changing conformation. The additive effect of these changes accounts for how the structural arrangement of the protein allows for the Ca²⁺ binding, the subsequent bioluminesence, and the slower rearrangement of certain helices in aequorin, yields the slower bioluminescence response (1).