Ca2+ binding enhanced mechanical stability of an archaeal crystallin

Abstract

Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and (10)FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a "mechanical clamp". Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to β-sandwich topology consisting of Greek key motifs but its overall structure lacks the "mechanical clamp" geometry at the termini. M-crystallin is a Ca(2+) binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens β and γ-crystallins. We constructed an octamer of crystallin, (M-crystallin)8, and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ∼ 90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and (10)FNIII, as well as lacking "mechanical clamp" geometry, can be mechanically resistant. Furthermore, Ca(2+) binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ∼ 35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca(2+) binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.

Figure 1. Structure and 2D topology diagram of two β-sandwich proteins with Greek key motifs used in this study.

The pulling direction used in the single-molecule force spectroscopy (SMFS) experiments is shown by arrows. (A) NMR structure of I27 (PDB ID: 1TIT). Terminal β-strands A′ and G are directly connected by H-bonding, shearing this “mechanical-clamp” results in the mechanical unfolding of the protein. The rupture of H-bonds between A and B strands constitutes the less stable mechanical intermediate. (B) 2D topology diagram of I27. The five-stranded (BCDEF) ‘double’ Greek key (3,2)₃ formed by overlapping (3,1)_N and (2,2)_C Greek keys (as defined by Hutchinson and Thornton [53]). (C) NMR structure of M-crystallin (PDB ID: 2K1W) bound to two Ca²⁺ ions (shown as black spheres). The terminal β-strands A and H are not directly bonded and they need to be “peeled” away from each other to unfold the protein. (D) 2D topology diagram of M-crystallin showing the two (3,1)_C Greek keys formed by ABCD and EFGH. In both cases, the backbone H-bonding around the terminal strands is shown.

Figure 2. Mechanical unfolding of (M-crystallin)₈ using SMFS.

(A) A pair of typical single-molecule force extension (FX) traces of apo protein (black). A series of equidistant force peaks in FX traces indicating the sequential unfolding of individual M-crystallin units in the octamer during the mechanical stretching (pulling speed is 1000 nm/s). The unfolding force peaks in sawtooth pattern are fitted with WLC model (grey). The contour length change is ∼29 nm and the unfolding force is ∼90 pN. Histograms of contour length change fitted to Gaussian distribution (B) and unfolding force (C) are shown.

Figure 3. Mechanical unfolding of Ca²⁺ bound (M-crystallin)₈.

(A) A pair of FX traces obtained in the presence of 10 mM Ca²⁺. The contour length change upon unfolding is ∼29 nm and the unfolding force is ∼125 pN in the sawtooth curves (black). WLC fits are also shown (grey). (B) The unfolding force histograms of (M-crystallin)₈ in holo (filled) and apo (unfilled) show that Ca²⁺ stabilizes the protein mechanically by ∼35 pN.

Figure 4. [Ca²⁺] dependent unfolding forces of (M-crystallin)₈.

The unfolding force histograms at various Ca²⁺ concentrations are shown in Figure S7 in File S1. The increase in unfolding force in two phases is consistent with two Ca²⁺ binding sites (see text for more details).

Figure 5. Pulling speed dependence on mechanical unfolding of (M-crystallin)₈.

A semi-logarithmic plot of unfolding force versus pulling speed for apo protein (○) and holo protein (•). Errors bars in the experimental data are SE. The Monte Carlo fits (solid line) are also shown for apo and holo proteins. Results from Monte Carlo simulations are given in Table 2. It is evident from the data that Ca²⁺ binding mechanically stabilizes M-crystallin by ∼30 pN at all pulling speeds.

Figure 6. A schematic of two-state energy level diagram depicting the thermodynamic and mechanical stabilization of M-crystallin upon Ca²⁺ binding.

Ligand binding not only stabilizes the native state (N) by ΔG∼11.4 kcal/mol but also reduces the unfolding potential width (Δx_u) from 0.55 nm to 0.38 nm. Estimates of unfolding transition state (TS) energy barriers (ΔG^‡) are also indicated. See text and Tables 1 and 2 for more details.