The tip of the dome is also the closest approach to the seven-fold symmetry axis in the Mt Cpn-10, where three acidic residues, D51, E52, and D53, are located (Fig. 1). The conglomeration of acidic residues, therefore, results in a very high negative potential towards the top of the Mt Cpn-10 dome (13, 14). The repulsive electrostatic forces thereby lead to an altered flexibility of the dome and possibly the instability of the oligomeric assembly (14). It is significant to note that Cpn-10 of E. coli has previously been proposed to have a metastable oligomeric structure leading to an increase in the diameter of the dome orifice in solution (15). The evolutionary conservation of flanking residues (residue positions 46-48 preceding the dome loop and residue positions 65 and 66 following the loop) and the charged residues at the tip of the loop (residue positions 51-53) suggest a possible critical role of the dome loop in the Mt Cpn-10 function (14). It has recently been shown that metal ion binding to the dome loop modulates the different plastic states of Mt Cpn-10 and enhances the stability of the oligomeric structure. It has also been proposed that the utilization of negative charges on the surface of the dome and their neutralization with metal ions enables the flexible dome loop to act as a ?Conformational Switch? for the Cpn-10-Cpn-60 interaction (14). In this work, we have explored the consequence of metal ion-binding at the dome loop, and whether the conformational changes in the dome loop due to metal-binding are effectively transmitted to different parts of the Mt Cpn-10 structure.

There have been several illuminating computational studies probing the dynamics of the Cpn-10-Cpn-60 complex (16, 17, 18), however, there has been no work reported to date examining the dynamics of Cpn-10 alone. Additionally, there is relatively little work done on the effects of metal ions on the dynamics/flexibility of proteins (19, 20). In the present study, we have carried out fluorescence experiments to probe the flexibility of the metal-binding regions of Mt Cpn-10 followed by molecular dynamics (MD) simulations of the protein. The MD simulations were carried out in an attempt to gain atomic level insights into the influence of metal ions upon the conformational changes in the protein.

Materials and Methods

Generation of the Dome Loop Mutant and Purification

The Mt Cpn-10 mutant, with the three acidic residues at the dome replaced by valine or alanine, was constructed using the Quik-Change site-directed mutagenesis kit (Stratagene). The forward and reverse primers for mutagenesis of D51V, E52A, and D53V were 5'-GGCCGGTGGGTCGCGGTCGGCGAGAAGCGG-3' and 5'-CCGCTTCTCGCCGACCGCGACCCACCGGCC-3', respectively, with the underlined nucleotides denoting the changed residues. Purification of the wild type and mutant protein was performed as previously described (14).

Assessment of Ca²⁺ Binding

Fluorescence emission of tryptophan as a function of changing Ca²⁺ ion concentration (100nM to 10mM) was used as a probe for conformational changes through equilibrium binding studies. The reaction mixtures containing 2μM (protomer) Cpn-10 in 10mM Tris-Cl, pH 8.0 were incubated with Ca²⁺ in a total reaction volume of 200μl at 25° C. The excitation wavelength was set at 295 nm and emission was measured in the range 310 and 500 nm. The equilibrium binding constant for the wild type Cpn-10 was determined by the increase in fluorescence upon ligand binding. Steady-state emission at each ligand concentration was monitored at 340nm and the observed fluorescence intensity corrected for the emission intensity of the reference buffer. Apparent binding constant (K_app) was determined by means of the following Equation [1] (21):

1/ΔF = 1/ΔF_max + 1/[K_app · F_max(c_p-c_o)]....................[1]
where ΔF is the change in fluorescence emission intensity at 340 nm (λ_ex = 295 nm) upon addition of different concentrations of Ca₂₊ and ΔF_max is the same parameter when the ligand is totally bound to the protein. c_p is the concentration of the protein and c_o is the initial concentration of the ligand. This equation is valid when c_o>>c_p. A linear plot of 1/ΔF against 1/(c_p-c_o) was extrapolated to the ordinate. The reciprocal of the intercept on the ordinate gave the value of ΔF_max. K_app was obtained from the ratio of the intercept and the slope of the plot.

Fluorescence polarization anisotropy of the wild type and the dome loop mutant Cpn-10 was monitored at varying concentrations of Ca²⁺. 2μM Cpn-10 in 10mM Tris-Cl, pH 8.0 was incubated with Ca²⁺, with concentrations of Ca²⁺ varying from 100nM to 10mM. Parallel and perpendicular emission components were measured by excitation at 295nm with a 5.5nm slit width. Emission was monitored through 8nm slit width at 340nm. In each anisotropy measurement, the parallel and perpendicular intensity of the background buffer solutions were subtracted from the measured values of the sample and the anisotropy was calculated. Measurements were repeated three times at each titration point and the resulting anisotropy values were averaged.

Fluorescence quenching experiments were performed with acrylamide as an external quencher. Acrylamide was added from a 1M stock solution to a final concentration ranging from 0.01M to 0.1M. Quenching was performed in the absence or presence of 1mM Ca²⁺ added to 2μM wild-type Cpn-10 in 100mM Tris-Cl, pH 8.0 at 25° C. The data were corrected for the dilution introduced by the addition of acrylamide. The fluorescence quenching data were analyzed according to the Stern-Volmer relationship [2] (22):

Fo/F = 1 + K_SV[Q]....................[2]
where Fo and F are the fluorescence intensities in the absence and presence of acrylamide, respectively. [Q] is the acrylamide concentration and K_SV is the quenching constant. The fraction of accessibility (fe) of the tryptophan residue was calculated from the Equation [3] (23, 24):

Fo/(Fo-F) = 1/(K_SVf_e[Q]) + 1/f_e...................[3]
Molecular Dynamics Simulations

Two MD simulations were carried out, one in the presence of calcium ions and the other in its absence (henceforth referred to as ION and NOION, respectively). The X-ray crystal structure of Mt Cpn-10 (8) (PDB code-1HX5) was taken as the starting structure for the study. Each monomer in the crystal structure has 97 amino acids with density for residues 1-4, 17-21, 31-36, and 98-99 missing. Hence, the numbering system used in our work will be from 1-93 in the subsequent text, with residues 1 and 93 corresponding to residues 5 and 97 in the crystal structure, respectively. While the terminal missing residues were left out, residues 17-21 and 31-36 were modeled on a monomer and the heptamer was generated accordingly. The models were constructed based upon the coordinates of the GroES:GroEL complex of E. coli (PDB code-1AON) (4). Ca²⁺ ions were modeled into each monomer on the basis of the position of the divalent ions observed in the crystal structure of Mt Cpn-10 (8). Ions were modeled in such a manner that each ion was stabilized by residues E9 and W50 of one monomer, and D61 of the neighboring subunit (residues 5, 46, and 57 in the renumbered system). The ions were placed at the positions of highest electronegativity, which roughly corresponded to the centre of the coordinates of the charged oxygens of the three residues. The ions were constrained during minimization but were free to move during the part of the simulations that was used for analysis.

The system was explicitly solvated with a shell of TIP3 water molecules (25) using the MOIL-View (26) program in the AMBER software suite. The number of TIP3 water molecules added to the system was 8735. Non-bonded electrostatic interactions, scaled by a constant dielectric value of 1.0, were shifted to zero at 12Å while a switching function was used to truncate the van der Waals interactions between 8-12Å; non-bonded lists were generated over 14Å. The simulations were carried out using the program CHARMM and its associated force field (27, 28). The conformation of the solvated protein system was first relaxed via a series of energy minimizations under harmonic constraints that were periodically reduced until zero. This was followed by MD simulations at 300K where the bonds to hydrogens were constrained using SHAKE (29) with a relative tolerance of 1.0 × 10^-6. The Leap Frog algorithm was used to calculate the Newton?s laws at time intervals of 1fs for a total of 2ns. Coordinates were saved every 1ps. The last 1.8ns of each simulation was used for analysis. The tools used for analyses included GROMACS (30, 31), VMD (32), SQUID (33), GRASP (34), Swiss PDB Viewer (35), MOLSCRIPT (36), and other locally written codes.

Results and Discussion

The plasticity of Mt Cpn-10 has been shown to arise due to the accumulation of acidic residues at the tip of the dome loop (Fig. 1). Moreover, addition of divalent cations has been shown to be effective in neutralizing the negative charges at the dome loop (14). In order to assess if the negative charges and the oligomeric plasticity were indeed correlated, a Mt Cpn-10 mutant, where the three acidic residues were replaced by valine or alanine, was constructed. Sequencing of the mutant gene confirmed that the mutant protein contained V, A, and V in place of D51, E52, and D53, respectively.

Conformational Changes Effected by Ca²⁺

The only tryptophan residue in the Mt Cpn-10 is present in the dome loop and lies at the monomer-monomer interface thereby serving as a probe for micro-conformational changes that occur upon ligand binding. In order to investigate interactions between Ca²⁺ and Mt Cpn-10, equilibrium binding was assessed through fluorescence intensity changes. As shown in Figure 2A, the emission intensity increased with increasing Ca²⁺ ion concentrations till it reached a saturation value at a concentration of 1mM in the wild type Cpn-10. The apparent binding constant thus calculated was 0.007μM^-1 (dissociation constant, K_d = 143μM) indicating a strong interaction between Ca²⁺ and the wild type Cpn-10. However, in contrast to the wild type, the emission intensity of the dome loop mutant upon variation of Ca²⁺ ion concentration remained relatively constant (Fig. 2B), thus suggesting that the mutant protein might not bind Ca²⁺.

Fluorescence anisotropy measurements provide a powerful means of assessing conformational changes, especially in cases like Mt Cpn-10, where the observations are due to a single tryptophan residue. Solvent exposure of W50 (W46 in the renumbered sequence) was measured through fluorescence anisotropy, which was thus exploited to probe conformational changes as a result of Ca²⁺ binding to the dome loop. Fluorescence anisotropy of tryptophan resulted in a profile as shown in Figure 2C. Binding of Ca²⁺ to the wild type Mt Cpn-10 resulted in a steady increase in fluorescence anisotropy indicating a decrease in the rotational freedom of the tryptophan, till saturation was achieved at 1mM. The dome loop mutant on the other hand did not exhibit any alteration in fluorescence anisotropy, corroborating our earlier data that the mutant does not exhibit Ca²⁺ dependent conformational changes. Thus, the dome loop mutant might have an impaired Ca²⁺ binding ability.