If we are to attempt to account for the intricacies of function of these photoproteins on the basis of their spatial structure, it is necessary to make a very detailed structural analysis and so a brief overview might be first useful to consider. There is very little gross structural difference among the different conformation states of the photoproteins, but closer examination reveals that there are local changes among the different structures as well as different intraprotein interactions between the aequorin and obelin. Probably most significant are the properties of the three calcium-binding loops. It is known that loop residue positions have to move in order to bind a Ca2+ in its typical coordination. It has been proposed that a loop should have a higher affinity for Ca2+ if the coordinating residues do not have to move much for accommodation of the Ca2+. It would follow that loop I belonging to EF-hand motif I should have a higher affinity than the loops in EF-hands III and IV, and these required position shifts and therefore the affinities differ between aequorin and obelin. However, the situation is quite complex because there are loop–loop H-bonds as well as EF-hand helix–helix interactions, so that the binding to one loop will influence the binding to the others. On the basis of a previously formulated hypothesis that a displacement of His175 in EF-hand IV of obelin was responsible for triggering the bioluminescence, a mechanism of transmission of the calcium signal is proposed.
Overall structure
Figure 2
shows that the structures of the Ca
2+-loaded apo-photoproteins (Fig. 1
, state
V), retain the same compact scaffold and characteristic two-domain fold as the undischarged photoproteins (state
II) (
Head et al. 2000;
Liu et al. 2000,
2003). Table 1
shows that the same conclusion can be made for comparison with the Ca
2+-discharged obelin (Fig. 1
, state
IV), which retains coelenteramide (
Deng et al. 2004b).
| Figure 2.Crystal structures of Ca2+-loaded apo-aequorin and apo-obelin and their comparison. (A) Cartoon drawing of the crystal structures of Ca2+-loaded apo-aequorin (state V). (B) The crystal structure of aequorin (state II, monomer B of the dimer; PDB code (more ...) |
| Table 1. RMS deviation values of different states of Ca2+-regulated photoproteins |
A calcium ion is found at each of the expected Ca2+-binding sites, EF-hand loops I, III, and IV, in both proteins (Fig. 2A,D ), and the N termini of both proteins are disordered. The C terminus of Ca2+-loaded apo-obelin is not observed in the electron density map and is assumed to be disordered as well.
Although Ca2+-loaded apo-aequorin and apo-obelin share a similar overall fold, easily discerned local structure differences are found for almost every part of the structures, both in the helical and the loop regions (Fig. 2E ). The RMSD of the Cα atoms of the two Ca2+-loaded apo-protein structures (from residue 15 to 181 of Ca2+-loaded apo-obelin) is 2.27 Å. But the RMSD of the same residue range of aequorin and obelin, for example, is only 1.48 Å. This means that the similarity in structure between the photoproteins is greater than between the Ca2+-loaded apo-proteins. The magnitude of this difference leads us to suggest that the different conformational transients following Ca2+ binding might be responsible for the differences in properties such as bioluminescence kinetics.
Structural comparison of different states of photoproteins
Table 1
is a collection of RMSDs of different conformation states. The overall RMSD for the aequorin states
V versus
II is 2.15 Å and for the same obelin states, 2.91 Å. It should be noted that for both proteins, the displacement of the C-terminal domain (containing EF-hand loops III, IV) is much bigger than that of the N-terminal domain (containing EF-hand loops I, II). Another finding is that there is hardly any difference in RMSD whether the Ca
2+-loaded apo-obelin is compared to either obelin (Fig. 1
, state
II) or the Ca
2+-discharged obelin (Fig. 1
, state
IV).
In both the aequorin and obelin structures (Fig. 1 , state II) the C terminus caps the substrate cavity containing the hydroperoxycoelenterazine, resulting in a solvent-inaccessible and nonpolar environment (Fig. 2B ). This condition apparently optimizes the efficient population of the first electronic excited state of coelenteramide and favors a high quantum yield for its fluorescence (Watkins and Campbell 1993). In the obelin case for state IV, the C terminus cap remains over the coelenteramide (Deng et al. 2004b), but its position cannot be detected in the Ca2+-loaded apo-obelin. In the Ca2+-loaded apo-aequorin structure, however, the C terminus is opened up. This was proposed by Kendall et al. (1996) as the reason apo-aequorin can have a luciferase function. Assuming that uncapping occurs in both photo-proteins going from state III to state IV, this would explain why the coelenteramide dissociates away in the presence of the organic solvent used for the crystallization, or in the case of Ca2+-discharged aequorin in solution, by removal of bound Ca2+ (Shimomura and Johnson 1970, 1975).
Another noticeable difference is that particularly in the case of aequorin going to the Ca2+-loaded apo-aequorin (state V), the structure becomes more elongated, especially the EF-hand motifs III and IV move closer toward each other, leaving no space in the cavity for coelenteramide.
Calcium-binding loops
Figures 3
–5
show how the 12 residues of the EF-hand calcium-binding loops I, III, and IV shift their positions when going to the Ca
2+-loaded states (Fig. 1
, state
V). The typical geometrical arrangement of oxygen atoms in a pentagonal bipyramid is observed with the Ca
2+ ion occupying the center of the pyramid. All three Ca
2+-binding sites of both apo-proteins (Figs. 3C,E
; 4B,D
; 5B,D
) contribute six oxygen ligands derived from the carboxylic side-chain groups of aspartate and glutamate residues, carbonyl groups of the peptide backbone or side chain of asparagine, and hydroxyl group of serine to the metal ion, with a separation of ~2.4 Å (Table S1). The seventh ligand comes from the oxygen atom of a water molecule. Based on studies of EF-hand calcium-binding proteins from different sources, it has been observed that high-affinity Ca
2+-binding sites have either no water or at most one water ligand (
Strynadka and James 1994). The three Ca
2+-binding sites of the present Ca
2+-loaded apo-photoprotein structures each contains only one water molecule as a ligand, indicating that they all can have high affinity for calcium.
| Figure 3.Structure of calcium-binding loop I in different photoprotein states. (A) Obelin (PDB code 1EL4; state II). (B) Ca2+-soaked obelin crystal (PDB code 1QV1). (C) Ca2+-loaded apo-obelin (state V). (D) Ca2+-discharged W92F obelin (PDB code 1S36; state IV (more ...) |
| Figure 5.Structure of calcium-binding loop IV in different photoprotein states. (A) Obelin (PDB code1EL4; state II). (B) Ca2+-loaded apo-obelin (state V). (C) Ca2+-discharged W92F obelin (PDB code 1S36; state IV). (D) Aequorin monomer A of the dimer (PDB code (more ...) |
| Figure 4.Structure of calcium-binding loop III in different photoprotein states. (A) Obelin (PDB code 1EL4; state II). (B) Ca2+-loaded apo-obelin (state V). (C) Aequorin monomer B of the dimer (monomers A and B are very similar) (PDB code 1EJ3; state II). (D) (more ...) |
Liu et al. (2003) reported that an obelin crystal that had been soaked with a very low concentration of Ca2+ such as not to trigger the bioluminescence, had a Ca2+ bound in loop I only. They concluded that loop I must have a higher affinity for Ca2+ than the other loops. In fact, the ligands in loop I more than in loops III and IV of obelin appear to be pre-positioned for the binding, requiring only small shifts to properly accommodate Ca2+ (Table 1; Fig. 3A–C ; Strynadka et al. 1997). It was also noted that the coordination was not in the usual pentagonal bipyramidal configuration as the invariant ligand Glu41 (Fig. 3B ) was absent, hardly shifted from its position in obelin. This Ca2+-soaked state in the crystal might represent an intermediate in calcium binding in solution (Fig. 3B ), going from the calcium-free state (Fig. 3A ) to the properly coordinated calcium-loaded state (Fig. 3C ). It can be expected that in solution there could be a dynamic equilibrium set up with the Glu41 able to move in to ligate Ca2+. Adding higher concentration of Ca2+ to the soaked crystal or prolongation of soaking with the same calcium concentration caused it to crack accompanied by bioluminescence emission.
Strynadka and James (1994) proposed that conformational “pre-forming” of the ligands in the binding loop would decrease an energetic (presumably entropic) cost in ordering this site for optimal calcium binding. As a consequence, the Ca2+-binding loop I should have higher affinity for calcium than Ca2+-binding loops III and IV. In other words, calcium ion will first go to the Ca2+-binding loop I. The residues in Ca2+-binding loop III are at close orientations but not quite the right positions for calcium binding (Fig. 4 ) and have to adjust a little to accommodate a calcium ion. The RMSD between Ca2+-loaded and Ca2+-free states (state V vs. state II) of the residues of this loop is higher than that of loop I (Table 1). Most displacement is also observed for the Glu at loop residue position 12. Therefore, on the assumption of the inverse relation of residue displacement to the affinity to calcium, loop III should bind calcium with less affinity than loop I.
Among the three Ca2+-binding loops of obelin, loop IV has to undergo the largest change in terms of residue reorientation and repositioning (Fig. 5 ; Table 1), and therefore the affinity of this Ca2+-binding loop may be less than that of the others. According to the hypothesis for the mechanism of triggering photoprotein bioluminescence suggested by us, the crucial step of bioluminescence initiation is a spatial displacement of His175 (Deng et al. 2004b; Vysotski and Lee 2004). The significant conformational change of Ca2+-binding loop IV to accommodate calcium supports this requirement because His175 is found in the exiting α-helix of this loop.
In the Ca2+-binding loop structure comparison of ae-quorin and Ca2+-loaded aequorin (Figs. 3 –5 ), it is easily seen that the loop regions of the EF-hand motifs I (Fig. 3E ) and III (Fig. 4C ) of aequorin do not have a geometry as optimized as has obelin for accommodating Ca2+. In ae-quorin a couple of residue side-chain ligands have to reorient, whereas in obelin, only the bidentate ligand Glu needs to move significantly. For the loop region of the EF-hand motif IV (Fig. 5 ), aequorin also needs to change a lot more than does obelin for Ca2+-binding. Aequorin was crystallized as a dimer (Head et al. 2000). The loop residue conformations of monomer A are more removed from the final Ca2+-occupied conformation than that of monomer B. It appears that structures of the Ca2+-binding loop residues of obelin and aequorin are different. The loop regions of obelin need less conformational changes than do those of aequorin for Ca2+ attachment; in other words, obelin is pre-positioned for Ca2+ binding more than aequorin. As a consequence, this could be the reason that obelin has a faster response to Ca2+ concentration change than does aequorin (Campbell 1974; Moisescu et al. 1975; Illarionov et al. 2000a).
Interactions between EF-hand motifs in N- and C-terminal domains
In the family of EF-hand proteins, the EF-hand motif almost always occurs in pairs. This double motif appears to be important for correct structural folding, and is thought to increase the affinity of each EF-hand for calcium (
Kretsinger 1980;
Kawasaki et al. 1998;
Nelson and Chazin 1998). The paired EF-hands display extensive hydrophobic interactions between the two EF-hand motifs, and a short β-type interaction between the two binding loops.
The EF-hand motif I of photoproteins is paired with EF-hand motif II, which has the characteristic structural features of an EF-hand motif but not the canonical sequence in the loop for calcium binding. The two loops of EF-hand motifs I and II show typical interactions in both obelin and aequorin (Fig. 1 , state II). In obelin, the loop I is bound with the loop II by means of hydrogen bonds between main-chain nitrogen and carbonyl oxygen atoms of Ile37 and Ile83, the side-chain Oδ1 of Asp40, and the main-chain nitrogen atom of Gly80, and between Nζ of Lys36 and the side-chain oxygen atom of Glu82 (Fig. S2). Aequorin displays exactly the same hydrogen-bond interaction pattern between these loops, even though a threonine in aequorin is found in a position corresponding to Ile83 in obelin without affecting the hydrogen-bond network.
In Ca2+-loaded apo-obelin and Ca2+-loaded apo-aequorin (Fig. 1 , state V), the hydrogen-bond pattern of loop–loop interaction is different. The binding of calcium ion in loop I abolishes the hydrogen bond between Lys36 and Glu82 of obelin and the same is observed for Ca2+-loaded apo-aequorin versus aequorin. In obelin the hydrogen bond distances between the main-chain nitrogen atom of Ile37 and the main-chain carbonyl oxygen atom of Ile83, and the main-chain carbonyl oxygen atom of Ile37 and the main-chain nitrogen atom of Ile83 are 3.03 Å and 2.80 Å, respectively; in the Ca2+-loaded state of apo-obelin, the distances are switched to 2.91 Å and 3.03 Å. The accommodation of calcium in the loop makes both distances of the corresponding interactions in Ca2+-loaded apo-aequorin shorter. The hydrogen bond distances between Oδ1 of Asp40 and the main-chain nitrogen atom of Gly80 that bind helices B and C of obelin, are practically unchanged, whereas an increase in distance on calcium binding occurs in the case of aequorin.
The loops of EF-hand motifs III and IV interact with each other as well both in Ca2+-free (Fig. 1 , state II) and in Ca2+-loaded states (Fig. 1 , state V) of obelin and aequorin. The interaction occurs mainly through hydrogen bonds between main-chain atoms of Ile and Leu in both photoproteins (Fig. S3). However, in the case of aequorin there is an additional hydrogen bond between the main-chain carbonyl oxygen atom of Gly122 and the main-chain nitrogen atom of Val162 (Fig. S3b). The accommodation of a calcium ion in these loops produces changes in hydrogen bonding between them with similar trends as observed in the N-terminal domain. In the Ca2+-free state (state II) of obelin the hydrogen bond distances between the main-chain nitrogen atom of Ile130 and the main-chain carbonyl oxygen atom of Leu166, and the main-chain carbonyl oxygen atom of Ile130 and the main-chain nitrogen atom of Leu166, are 2.89 Å and 3.01 Å, respectively (Fig. S3a). In the Ca2+-loaded state (state V) the distances are switched to 3.09 Å and 2.84 Å (Fig. S3c). The corresponding distances in aequorin become shorter in the Ca2+-loaded state (state V) of apo-aequorin (Fig. S3b,d). The hydrogen bonding between Gly128 and Val162 in aequorin is abolished after Ca2+ binding (Fig. S3b,d).
These observations allow the reasonable assumption that these two photoproteins may undergo different conformational transients of the Ca2+-binding loops following the binding of calcium, an additional possible origin for the observed differences in bioluminescence kinetics.
Interhelical angles and interaction of the α-helices
The conformational changes that result from calcium binding to loops of the EF-hand motifs are usually described by the interhelical angles that represent more complex rearrangements of the interfaces between the α-helices of the domains. Although such a description of Ca
2+-induced conformational transients is available only for a few Ca
2+-binding proteins, it provides a useful framework within which different types of conformational changes can be examined. For instance, the EF-hand motifs of calmodulin and troponin C are approximately antiparallel in the Ca
2+-free state, but they become nearly perpendicular in the Ca
2+-loaded state. This reorientation of α-helices exposes a hydrophobic patch, which is the basis of the biological function of these two proteins in living cells.
Unlike calmodulin or troponin C, the interhelical angles in both obelin and aequorin are almost perpendicular already in their Ca2+-free state (Fig. 1 , state II), and the Ca2+ binding does not change these angles significantly (Table 2; Fig. S4). In comparison to the effects in calmodulin and troponin C, we can conclude that the α-helices of Ca2+-regulated photoproteins in state II (Fig. 1 ) are optimally oriented for calcium binding. This also is in agreement with our conclusion derived from analysis of the orientation of the residues of the Ca2+-binding loops. As a consequence of the only small conformational shifts that are required to trigger the bioluminescence, these photoproteins are able to respond to the appearance of Ca2+ on the millisecond time-scale.
| Table 2. Interhelical angles in EF-hand motifs of different states of Ca2+-regulated photoproteins (degrees) |
Although these changes are observed for the Ca2+-loaded apo-states (Fig. 1 , state V) of photoproteins, we suppose that similar displacements of the α-helices will occur when the coelenteramide is bound (Fig. 1 , state III) because, for instance, the presence of coelenteramide in Ca2+-discharged obelin (state IV) does not drastically affect the orientation of α-helices as compared with obelin itself (Table 2).
A much-debated question regarding the bioluminescence of Ca2+-regulated photoproteins is the precise series of molecular events produced by calcium binding to the protein that results in triggering the bioluminescence. As just mentioned, an obelin crystal soaked with a low concentration of Ca2+ produced a spatial structure in which one calcium ion occupied the Ca2+-binding loop I (Liu et al. 2003). Therefore, the first molecular event in solution is the calcium binding with the Ca2+-binding loop I, and any rearrangement of α-helices A and B caused by this binding.
The α-helices in these photoproteins exhibit many interactions, van der Waals, ion pairing, and hydrogen bonds. Especially there are many hydrogen bonds between helix A and helix H, and it should be again noted that His175, crucial for the bioluminescence triggering (Deng et al. 2004b; Vysotski and Lee 2004), is found in the exiting helix H before the C terminus of the protein. This hydrogen-bond connectivity is strictly conserved between obelin and aequorin. Figure 6 shows the hydrogen-bond interhelical network for the Ca2+-free (state II) and Ca2+-loaded states (state V) of obelin and aequorin. In the Ca2+-free state, hydrogen bonds from the Arg to His are found in both proteins (Fig. 6A,B ). The Arg hydrogen bond binds together helix A and the C terminus that caps the substrate cavity containing the hydroperoxycoelenterazine, resulting in a solvent-inaccessible and nonpolar environment. The hydrogen bonds His–Trp and Arg–Phe probably also serve this goal pulling together helix A and helix H. In Ca2+-free aequorin (state II) there are also numerous additional hydrogen bonds that bind these two α-helices and also helix A to the C terminus but are lacking in obelin and probably produce a more stable interaction in the case of aequorin.
| Figure 6.Hydrogen-bond interactions between helix A and helix H, and helix A and the C terminus in conformation states II and V. (A) Obelin (PDB code1EL4; state II). (B) Aequorin (PDB code 1EJ3, monomer B; state II). (C) Ca2+-loaded apo-obelin (state V). (D) Ca (more ...) |
In the Ca2+-loaded states (state V) of apo-obelin (Fig. 6C ) and apo-aequorin (Fig. 6D ), the hydrogen-bond network is completely different compared to the Ca2+-free states (state II). In the case of Ca2+-loaded apo-obelin (state V), the hydrogen bonds binding together helices A and H are gone and there are only two hydrogen bonds remaining between Arg21 and Arg17 and the residues from the C terminus. This clearly shows that helix A and helix H move away from each other upon calcium binding. Different changes are observed for Ca2+-loaded apo-aequorin. In this case, despite the fact that the hydrogen-bond pattern is also different for the Ca2+-loaded state (state V) from in the Ca2+-free state (state II), helix A and helix H remain connected by numerous hydrogen bonds. 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 clearly implies that these photoproteins undergo different conformational transients on calcium binding.
In contrast to helix A, helix B forms only one hydrogen-bond interaction with helix C in both aequorin and obelin. Therefore, the probability of calcium signal transmission from the EF-hand motif I to other parts of the photoprotein molecule through changes of helix A is higher than through changes in helix B. As already mentioned, although calcium-induced conformational changes are represented by big changes in interhelical angles on calcium binding in some proteins, this is not the case for the Ca2+-regulated photoproteins (Table 2; Fig. S4).
Figure 7 shows the hydrogen-bond structure of helix A in obelin (state II), Ca2+-loaded apo-obelin (state V), aequorin (state II), and Ca2+-loaded aequorin (state V). For clarity, only the main-chain atoms are shown. The intrahelical hydrogen-bond connectivity of helix A in the Ca2+-free state (state II) of obelin is typical for an α-helical structure with the hydrogen bond distances indicated for each carbonyl to the fourth following residue. Although in general the intrahelical hydrogen bonds of helix A are maintained in the Ca2+-loaded state (state V) of apo-obelin, there is a small increase of 0.1–0.3 Å in each bond length (Fig. 7B ), which accumulates to a significant stretching of the helix length. In other words, the accommodation of calcium ion in the Ca2+-binding loop of the EF-hand motif I makes the incoming helix A less stable, and this can also be imagined as a pulling of helix A in the direction of the N terminus.
| Figure 7.Stereo view of helix A in conformation states II and V. (A) Obelin (PDB code1EL4; state II). (B) Ca2+-loaded apo-obelin (state V). (C) Aequorin (PDB code 1EJ3, monomer B; state II). (D) Ca2+-loaded apo-aequorin (state V). The photoprotein conformation (more ...) |
The intrahelical hydrogen-bond patterns of helix A for aequorin (Fig. 7C ) and obelin (Fig. 7A ) are almost the same. However, on going to the Ca2+-bound state (state V), the changes are completely different in that for aequorin, there appears to be some compaction of the incoming helix A.
Despite the difference in character of these changes in the two photoproteins, in both cases a helix A displacement will be transmitted to helix H via their hydrogen-bond interactions.
Intrastructural transmission of the calcium signal
According to our hypothesis (
Deng et al. 2004b;
Vysotski and Lee 2004), displacement of His175 that lies in helix H is a crucial step for triggering the bioluminescence. We first considered that binding of one Ca
2+ to the loop IV preceding helix H, and needing a significant repositioning of the coordinating residues to properly accommodate the Ca
2+, would propagate to a repositioning of His175. In the analysis above we have enumerated many hydrogen-bonding interactions among the EF-hand motifs, and no doubt other types of interaction must be present as well. In other words, we expect that binding to each of the loops will not be independent.
The likelihood that each binding event will trigger a bioluminescence response depends on three factors: the on-rate for Ca2+ binding, which is probably proportional to the binding affinity; the degree of residue shift for Ca2+ ligation and the rate of propagation of this change to helix H; and the amount of any position shift that His175 undergoes as a result. There are also hydrogen-bond interactions between loops III and IV, so that binding to the one should no doubt alter the positions of the key binding residues in the other.
Therefore, at least for obelin the sequence of molecular events on calcium binding leading to the triggering of bioluminescence could be as follows:
- A calcium ion is preferentially bound to loop I as evident from the observations of the soaked obelin structure and expected from the “pre-formed” nature of this loop itself.
- The binding of calcium ion to this loop and optimization of the pentagonal, bipyramidal geometry produces a “twist” of the EF-hand motif I around a pivot point by means of changes in hydrogen bond distances between the main-chain atoms of Ile37 and Ile83. The accommodation of calcium ion also changes slightly the interhelical angle between helices A and B.
- All these changes produce a pulling of helix A in the direction of the N terminus of the protein.
- Since helix A is tightly bound with helix H and the C terminus through numerous hydrogen bonds, the changes in helix A will result in a displacement of helix H and the C terminus. In other words, the binding of only one calcium ion into the Ca2+-binding loop of the EF-hand motif I could be sufficient to trigger the bioluminescence.
- The binding of a calcium ion to loop IV and the optimization of the pentagonal, bipyramidal geometry produces a “twist” of the EF-hand motif IV around a pivot point by means of changes in hydrogen bond distances between the main-chain atoms of Ile130 and Leu166. At the same time, the accommodation of calcium ion induces a small change of interhelical angle between helices H and G.
- The displacement of helix G produces a rearrangement of helix F, which is hydrogen-bonded with helix G. The displacement of helix F can therefore adjust the Ca2+-binding loop of the EF-hand motif III increasing its affinity for calcium and facilitating its binding. The accommodation of calcium into this Ca2+-binding loop completes the rearrangements of α-helices F and E of the EF-hand motif III and again leads to an additional stimulation of the bioluminescence. In fact, the accommodation of the third calcium ion and the rearrangements in the EF-hand motif III complete all the structural rearrangements in the photoprotein molecule, producing a final conformation that is optimal for effective bioluminescence.
Although we have considered in detail only the sequence of molecular events for obelin, we assume that in the case of aequorin, the “calcium signal transmission” through the photoprotein structure should be in general the same. The minor distinctions could probably serve as a molecular basis for the reported differences in bioluminescence properties. For instance, if the energy cost for the helicity increase of aequorin helix A is higher than for pulling obelin helix A, then it might result in a slower rearrangement of helix H in aequorin. Therefore, a slower bioluminescence response on calcium addition will occur for aequorin than for obelin as is observed (Illarionov et al. 2000a).
Based on aequorin bioluminescence titration with Ca2+ it has been proposed (Shimomura 1995; Shimomura and Inouye 1996) that only two Ca2+ ions are required for light emission and that the third Ca2+ is unrelated. Other authors using various experimental approaches with both obelin and aequorin have concluded that three Ca2+ ions were necessary (Moisescu et al. 1975; Allen et al. 1977; Campbell et al. 1979; Illarionov et al. 2000a; Markova et al. 2002). The sequence of molecular events occurring in a photoprotein molecule suggested by us based on structural data and the mechanism for triggering the bioluminescence (Deng et al. 2004b; Vysotski and Lee 2004) implies that binding of even one calcium ion into Ca2+-binding loop I will be enough to set off bioluminescence. The binding of the other two calcium ions is a cooperative event leading to a greater stimulation of bioluminescence, especially the binding of the second calcium ion to the Ca2+-binding loop of EF-hand motif IV. Our recommendation is that in view of the different affinities of the binding sites and probably cooperativity among them, one must interpret bioluminescence kinetic results with due caution.
Another study relevant to this debate was an investigation of obelin mutants with substitutions of key residues in each of the loops participating in calcium coordination in the Ca2+-binding loops (Illarionov et al. 2000b). The authors consecutively “switched off” the Ca2+-binding loops of obelin and studied some properties of these mutants. As a result of this investigation they concluded that all calcium-binding loops are necessary for effective bioluminescence. The structural data in the present work lead to the same conclusion but also allow us to account for the results obtained for obelin mutants from a structural point of view. The inter-helical angles of both obelin and aequorin are almost perpendicular already in the Ca2+-free state (Fig. 1 , state II), and therefore we concluded that all EF-hand motifs of Ca2+-regulated photoproteins are already pre-formed to bind calcium ion. The same conclusion derives from analyses of orientation of the key residues participating in calcium binding of the Ca2+-binding loops. Therefore, if even some Ca2+-binding loop(s) were mutated and did not bind calcium ion, the nonmutated loop(s) would probably do it even with the same affinity. Since the α-helices are all interacting with each other by hydrogen bonds, even the binding of one calcium into one binding loop could produce a displacement of helix H, but the path of calcium signal transmission in each mutant would be different in comparison with wild-type photoproteins, and therefore there will be a different effect on some bioluminescent properties of the mutated photoproteins such as bioluminescence response kinetics or the calcium concentration–effect curve, as examples, and, indeed, this is what is observed (Illarionov et al. 2000b).