Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd2+/Mg2+–con-T[K7γ] complex
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- Cnudde, S.E., Prorok, M., Castellino, F.J. et al. J Biol Inorg Chem (2010) 15: 667. doi:10.1007/s00775-010-0633-2
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Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in γ-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-d-aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca2+ and Mg2+ can fulfill this role, Ca2+ induces dimerization of con-G, whereas the Mg2+-complexed peptide remains monomeric. A variant of con-T, con-T[K7γ] (γ is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca2+ ions and two Mg2+ ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca2+ for dimer formation, we report here the structure of the monomeric Cd2+/Mg2+–con-T[K7γ] complex, and, by comparison with the previously published con-T[K7γ]/Ca2+ dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.
KeywordsCrystallographyConantokinsγ-Carboxyglutamic acidPeptide α-helix
The conantokins are a small family of γ-carboxyglutamate (Gla)-containing Conus peptides that act as potent and specific antagonists of the N-methyl-d-aspartate receptor (NMDAR). The most well-characterized members of this group include conantokin-G (con-G) and conantokin-T (con-T), which are 17 and 21 amino acids in length, respectively [1, 2]. The peptides share sequence identity within their first four residues and contain Gla residues at positions 3, 4, 10, and 14. Despite high overall homology, a conspicuous primary structural difference between the peptides occurs at sequence position 7. A Gla residue in con-G and a Lys residue in con-T occupy this locus. Their high relative contents of Gla residues permit con-G and con-T to interact with a variety of metal cations, which in turn contribute to the stabilization of their α-helical conformations. However, in the absence of metal cations, con-G adopts a random-coil structure devoid of secondary structural elements, whereas con-T assumes a high degree of helicity [3, 4]. These different secondary structural tendencies primarily derive from the nature of the residue at position 7, which provides for a destabilizing array of Gla residues spaced at i, i + 4, i + 7 intervals in con-G (positions 3, 7, and 10) in contrast to a stabilizing Gla3, Lys7, Gla10 arrangement of the corresponding residues in con-T. More recently, a series of solution studies revealed that the ability of con-G to undergo Ca2+-mediated antiparallel dimerization, a property not shared by con-T, is ascribable to the presence of Gla7 . This self-association has been demonstrated to rely on optimal intra- and interpeptide Gla–Ca2+ contacts that require an i, i + 4, i + 7, i + 11 motif of Gla spacing within the monomer. In support of this model, it has been shown that replacement of Lys7 in con-T with a Gla residue results in a peptide that can form an antiparallel dimer in the presence of Ca2+ . High-resolution crystal structures of the Ca2+-bound con-G and con-T[K7γ] (where γ is Gla) antiparallel dimers have been determined and confirm the predictions arising from the solution studies . Though the helices of con-G and con-T[K7γ] are virtually identical in these structures, as are the Ca2+ binding sites, the dimer interface is completely different. Paradoxically, both peptides have higher affinity for Mg2+ than for Ca2+  but neither dimerizes in the presence of Mg2+. Also, we have previously demonstrated that both the monomeric and the dimeric forms of the conantokins (as shown for con-G) display NMDAR activity, albeit with different kinetic signatures for each of the four NMDAR subunit combinations tested . In an effort to understand the apparent incongruity between metal affinity and dimerization, and to ascribe a molecular basis for the different NMDAR activity profiles of the monomer and dimer forms of the peptide toxin, a comparison of conantokin structures in monomeric and dimeric metal-bound states is required. Furthermore, insight into the interactions that prevail in the metal ion triggered self-assembly of peptide helices has applications across the biochemical spectrum, including metal sensoring and protein design [10–13]. With these objectives in mind, we have determined the X-ray crystallographic structure of the Cd2+/Mg2+–con-T[K7γ] complex, the first crystal structure of a monomeric conantokin, which is the physiologically relevant oligomerization state for con-T. The details of this structure and the salient differences that exist in the Ca2+-coordinated versus the Mg2+-coordinated species, with emphasis on the disposition of Gla residues, are reported herein.
Materials and methods
Peptide synthesis, purification, and characterization
Con-T[K7γ] was synthesized, purified, and characterized as previously described . The amino acid sequences of the peptides relevant to this communication, as well as their α-helical heptad repeat assignments, are provided later.
Lyophilized con-T[K7γ] was dissolved in 0.05 M MgCl2 and 100 mM tris(hydroxymethyl)aminomethane–HCl, pH 8.0, to a concentration of 10 mg/ml. Crystals were grown at 20 °C by the hanging drop vapor diffusion method in 1 M sodium acetate, 0.1 M N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid, pH 7.8, and 0.05 M CdSO4. The crystals appeared after 3 days.
Data collection and refinement statistics for the Cd2+/Mg2+–con-T[K7γ] complex
a, b, c (Å)
57.1, 57.1, 32.8
No. of measured reflections
No. of unique reflections
No. of atoms
Mean B factors
Bond lengths (Å)
The phases were determined by molecular replacement using the program Phaser  and the Ca2+–con-T[K7γ] structure as search model (Protein Data Bank ID 2DPR). Using one molecule per asymmetric unit , we calculated a Matthews coefficient of 2.2 Å/Da and solvent content of 43.8%, consistent with the existence of one molecule per asymmetric unit . The rotational and translational Z scores for the solution were 9.4 and 10.4, respectively, and the log likelihood gain (a measure of how good the model is relative to a random atom distribution) was 150.
Refinement of con-T[K7γ]
Isothermal titration calorimetry
Binding isotherms accompanying the titration of con-T[K7γ] with MgCl2 or CdCl2 were obtained by Isothermal titration calorimetry (ITC) using a VP-ITC calorimeter (MicroCal, Northampton, MA, USA). Peptide (0.1–0.2 mM) was dissolved in 20 mM 2-morpholinoethanesulfonic acid, 100 mM NaCl, pH 6.5, and placed over a small amount of Chelex-100 resin to ensure a metal-free species prior to the introduction of titrant. Titrations were performed at 25 °C with MgCl2 or CdCl2 (5–10 mM) dissolved in the above-mentioned buffer. The power compensations thus obtained were integrated over time and the resultant heat changes were deconvoluted for the best-fit model using the ORIGIN software program supplied by MicroCal. The parameters (n, Kd, ΔH) thus obtained represent the average of at least two experiments. Error estimates were 8% or less for replicate experiments.
Sedimentation equilibrium experiments were performed using an Optima XL-I analytical ultracentrifuge (Beckman Instruments, Palo Alto, CA, USA) in an An-60 Ti rotor equipped with a standard two-channel cell. Con-T[K7γ] was dissolved in 20 mM 2-morpholinoethanesulfonic acid, 100 mM NaCl buffer, pH 6.5, at a concentration of 0.20 mM. The peptide samples in the absence and presence of metal ions were independently rotated at 48,000 and 52,000 rpm at 20 °C until equilibrium was attained. Absorbance monitoring was performed at 275 nm. The apparent molecular weight (Mw,app) was obtained by global fitting of multiple scans (three to five scans at each speed from two separate analyses) to a single ideal species using the sedimentation analysis software supplied by Beckman. A partial specific volume of 0.711 ml/g was assigned to con-T[K7γ] . Gla residues were assigned the partial specific volume value of glutamate (0.66 ml/g). The fractional dimer content of self-associating peptides at equilibrium was calculated as previously described .
Structure of Cd2+/Mg2+–con-T[K7γ]
It is likely that Cd2+ is essential for crystallization, since no crystals grew in the absence of Cd2+, or even in decreased concentrations of Cd2+, consistent with the fact that three out of the four Cd2+ ions reside on crystallographic axes. It was not possible to determine whether the partially occupied Cd2+ sites are also partially occupied by Mg2+, since Mg2+ is significantly less electron dense than Cd2+ (46 vs. ten electrons). However, it is unlikely that Mg2+ is present at Cd3 because it is tetrahedral and Mg2+ is almost always octahedrally coordinated. The relatively hard Mg2+ cation is also not likely to coordinate nitrogen amines tightly. Further, the coordination lengths in the Cd3, Cd4, and Cd5 sites are significantly longer than those seen for Mg2+ (2.2–2.6 vs. 1.7–2.2 Å for a typical Mg2+). It would thus appear that the the Cd3, Cd4, and Cd5 sites occupied exclusively by Cd2+ and are critical for defining the crystal lattice contacts.
Comparison of Ca2+–con-T[K7γ] and Cd2+/Mg2+–con-T[K7γ]
Metal ion binding properties of con-T[K7γ]
Solution-phase molecularity of con-T[K7γ] in complex with Cd2+and Mg2+
Identity and specificity of the metal ion binding sites
Several questions become apparent from the data presented so far: (1) Does the Cd2+ dimer share the same dimerization interface as the Ca2+ dimer? (2) Which metal binding sites constitute the “tight” binding Cd2+ sites versus the weaker sites? (3) Are the tighter or the weaker Cd2+ binding sites involved in dimerization? (4) Why is the Mg2+-bound species monomeric, whereas the Cd2+ and Ca2+ species are dimeric?
The crystal structure of the Mg2+/Cd2+-bound peptide clearly identifies the two Mg2+ binding sites as being the Mg/Cd1 and Mg2 sites. The coordination distances in the remainder of the sites are not consistent with Mg2+ binding, whereas the coordination distances in both the Mg/Cd1 and the Mg2 sites are completely consistent with Mg2+ binding. It is thus clear that Mg2+ binds exclusively to the metal 1 and metal 2 binding sites, consistent with the 2:1 Mg to peptide stoichiometry found by ITC. It is also clear that at least one of the two Mg binding sites also defines the dimerization interface in the Cd2+-bound dimer. This is shown by the Mg2+ analytical ultracentrifugation (AUC) challenge experiments, which demonstrate that addition of Mg2+ leads to dimer dissociation. Therefore, Mg2+ binding disrupts the Cd-induced dimer, meaning that the dimer interface must include at least one of the Mg2+ ion binding sites. Further, the fact that dimer dissociation occurs only when the Mg2+ to Cd2+ ratio is very high suggests that the Mg2+ binding site or the site responsible for Cd2+ dimerization must overlap with at least one of the tight Cd binding sites, as opposed to the lower-affinity sites. This is because the loose Cd2+ binding sites have affinities around tenfold lower than those of the Mg binding sites. Therefore, even stoichiometric concentrations of Mg2+ would be expected to effectively compete for the weak Cd binding sites. The fact that it requires Mg concentrations tenfold or higher to begin to disrupt dimerization means that the Cd sites that are coincident with the Mg sites must be the high-affinity Cd binding sites. This is structurally consistent as these sites have higher coordination between the peptide and metal than do the other three Cd sites (both the metal 1 and metal 2 binding sites are tetracoordinate with the peptide). It is thus clear that the Cd and Ca dimer interfaces share at least one of the metal binding sites and are therefore likely to at least partially overlap.
The crystal packing in the structure is also instructive regarding this issue. Each con-T[K7γ] helix makes metal-lattice interactions with three different symmetry mates. This is in contrast to the Ca2+-bound con-G and con-T[K7γ] dimer structures, where all of the metal coordination was between one other helix partner, making it clear that the crystalline dimer was unambiguously the dimer found in solution. Although it is not possible to rule out the probability that some of the lattice interactions seen in our structure also occur in the Cd2+-bound solution dimer, it is absolutely clear that the crystal packing we see is not fully capitulated in solution as there is no evidence of such a large oligomer in solution. It is also telling that Mg2+ binding abrogates the solution dimer and the Mg2+ binding site is the only metal binding site that does not make lattice interactions.
Magnesium-bound monomers versus calcium-bound dimers
The question of why the Mg-bound peptide does not dimerize is explained by examination of the structures of Cd2+/Mg2+-bound and Ca2+-bound con-T[K7γ]. Sedimentation equilibrium data indicate that whereas the exclusively Ca2+- or Cd2+-complexed peptide is dimeric, the Mg2+-bound species is monomeric. In solutions containing both Cd2+ and Mg2+, the Mw,app of con-T[K7γ] decreases with increasing Mg2+ content. Solution-phase Mg2+-mediated dimer collapse has also been observed with Ca2+-saturated peptide .
From the crystal structure presented herein, it appears that Mg2+ occupancy of the metal ion site bridged by Gla10 and Gla14 is sufficient to disrupt the dimer; although there is also partial Mg2+ occupancy of the Cd1 locus maintained by Gla3 and Gla7. Because this site is only 50% occupied by Cd2+ and the coordination distances are substantially shorter than those found for the three other Cd sites, the Cd1/Mg1 site is likely to be partially occupied by Mg2+. However, it is apparent that the Gla10/Gla14 site, which has 100% Mg2+ occupancy, is the preferred Mg2+ binding locus. Although the model providing the best fit for our calorimetric data corresponded to two identical Mg2+ sites, the Kd value for Mg2+ binding (7.3 μM) could likely reflect an average value for two nonidentical sites that differ in affinity by only twofold or threefold, with the Gla10/Gla14 locus being the preferred Mg2+ site. This Mg2 site is similar to the Ca2 site in the Ca2+-bound con-T[K7γ] dimer, wherein Ca2 at this Gla10/Gla14 site chelates Gla residues 7 and 10 of the partner helix. The Cd2 site, which may have up to 50% Mg2+ occupancy, involves coordination to Gla 3 and Gla7. In the Ca2+–con-T[K7γ] structure, this locus corresponds to Ca1 and chelates Gla14 of the partner helix. Hence, Mg2+ occupies the same con-T[K7γ] sites that are occupied by Ca2+, but cannot induce formation of the dimer. This can be ascribed to the inability of Mg2+ to participate in the cross-helix interactions made by Ca1 and Ca2 in the Ca2+-bound dimer. As shown in Fig. 3b, the distance between Mg/Cd1 and Ca1 is about 0.84 Å. Ca1 coordinates with Gla10 and Gla14 of one helix and Gla7 and Gla10 of the dimer helix, whereas Ca/Mg1 only coordinates with Gla10 and Gla14. Given this placement, Mg1 is positioned 3.08 and 2.91 Å away from Gla10 and Gla7, respectively, of the partner helix of the Ca2+ dimer. These distances are too great to be spanned by Mg2+, which generally chelates with distances between 1.7 and 2.2 Å, whereas Ca2+ coordinates in a range from 2.2 to 2.6 Å. It is likely that if the helices were brought close enough together to accommodate Mg2+ coordination distances, the result would be unacceptable steric and electrostatic repulsion. Such prohibitively long interhelical distance considerations could also apply to Mg2+ binding at its putative second locus, i.e., the Mg/Cd1 site, in which Gla3 and Gla7 are involved in malonate-type chelation of the metal ion (Fig. 3c). Hence, despite the determination, by sedimentation equilibrium, that Cd2+ alone can induce dimerization of con-T[K7γ], the presence of Mg2+ at the metal ion binding site spanned by Gla10/Gla14 (and possibly Gla3/Gla7) is sufficient to destabilize the dimer, even though Cd2+ remains bound at other loci. However, given the high binding affinity for Cd2+ in the Mg2+ binding sites (0.2 vs. 7.2 μM for Cd2+ and Mg2+, respectively), it seems inconsistent that the crystal structure shows exclusive Mg2+ binding in one of the high-affinity metal binding sites and significant binding in the other. There are many potential explanations for this phenomenon but the simplest is that the present crystal form is consistent only with the monomeric peptide. Therefore, even though the Mg2+-bound monomer may have been the minor form in solution, it could still have been the form that crystallized, especially if its solubility is significantly lower than that of the dimer. The equilibrium would then be driven to produce the monomer in its solid form.
In previous work, we showed that monomeric con-G and a covalently constrained antiparallel dimer form of con-G are biologically active insofar as they both exert in vitro effects on the NMDAR . In that study, four physiologically prevalent subunit combinations of the NMDAR were utilized, namely, NR1a/NR2A, NR1a/NR2B, NR1b/NR2A, and NR1b/NR2B. Real-time electrophysiology traces revealed sharp differences in the mode of receptor activity exerted by each peptide species at the separate NMDAR combinations (e.g., the monomer is inactive at NR1a/NR2A-containing receptors, whereas the dimer potentiates at this combination), as well the kinetics of peptide action (e.g., the monomer shows slow onset and offset of inhibition at NR1b/NR2B, whereas the dimer inhibits with very fast onset and offset rates). These results suggest that distinctly different NMDAR activity profiles can be exhibited by a single conantokin, depending on the monomer–dimer distribution of the peptide in the vicinity of its NMDAR binding site. A possible reason for the differences in NMDAR activity between the monomer and the dimer can be culled from a comparison of Cd2+/Mg2+-bound and Ca2+-bound con-T[K7γ] structures (Fig. 3a), which reveals a significant reorientation of Gla4 between monomer and dimer states. This residue, which lies on the outer face of the dimeric species, is completely conserved in all ten conantokins characterized to date [19–21] and has been shown to be an absolute requirement for activity in con-G, con-T, and conantokin-R . We submit that the NMDAR subunit selectivity of a conantokin may be influenced by the disposition of Gla4, which, in turn, may be affected by the dimerization state of the peptide.
In conclusion, the structural analyses presented here underscore the importance of metal ion coordination geometry in influencing the molecularity of con-T[K7γ]. Additionally, these results raise the possibility that metal ion binding motifs that mimic the Gla10/Gla14 site can be engineered into other peptides or proteins in an effort to control oligomerization states in a reversible manner. Our data also show that a dimer similar to that formed by Ca2+ coordination can be formed with the much higher affinity Cd metal ion, meaning that much stronger metallozipper helical dimers can be formed than is possible using Ca.
We thank the following for financial support: GR-179 (085P100549) from the Michigan Economic Development Corporation (to J.H.G.), NIH Grant HL019982 (to F.J.C.), and NIH Grant GM0638947 (to J.H.G.). CCDC-667723 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.