The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

Abstract

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

Figure 1. Wt-azurin unfolds through two mechanically labile intermediates.

(a) Schematic structure of wt-azurin (PDB: 4AZU). (b) The residues trapped after the disulfide bond (yellow) are marked in white. The rest are represented in orange. (c) Scheme of the engineered (Azu–I27)₄ polyprotein being pulled in the AFM set-up. (d) Pulling on an individual (Azu-I27)₄ polyprotein results in an unfolding trajectory, where the unfolding of azurin (orange) occurs at ∼55 pN, followed by the unfolding of the I27 domains, distinguished by an unfolding force of ∼210 pN and an increment in contour length of ΔL=28 nm (purple). Inset: The unfolding of wt-azurin occurs via two well-defined mechanically stable intermediates occurring at ∼6 nm (green dashed line) or ∼9 nm (brown dashed line). (e) In a few trajectories (n=25), a four-state unfolding pathway is measured, where both intermediates (∼6 and ∼9 nm) are captured within the same trajectory. (f,g) Worm-like chain fits to the data reveal that each complete azurin unfolding event occurs concomitant to an increment in contour length of ΔL=38.6±0.9 nm, n=93 (f, orange histogram), requiring an unfolding force of 56.3±12.1 pN, n=93 (g, orange). When unfolding occurs via a mechanical intermediate, the main unfolding event (dashed lines), occurring at 54.1±11.3 pN, n=323 (g, grey), elicits a protein length of <10 nm (f, grey histogram) before it reaches a mechanically resistant intermediate. The mechanical disruption of such intermediate form occurs at forces of ∼42.3±12.3 pN, n=289 (g, blue), with a concomitant protein elongation of ΔL∼30 nm (f, blue histogram).

Figure 2. The Azu_C112A and the Azu_C26A mutations confirm the existence of parallel unfolding pathways.

(a) The Azu_C112A mutation prevents the formation of the Cu–S_Cys112 bond. (b) On unfolding, the Azu_C112A protein extends by ∼9 nm (dashed brown fit) before encountering a mechanical intermediate that corresponds to the disruption of the Cu–N_His46 bond (dark blue fit). (c) The C26A mutation prevents the formation of the disulfide bond. In the absence of the disulfide bond, each azurin unfolding event occurs concomitant to an increase in contour length of ΔL=47.6±1.2 nm, n=77 (orange fits). The mechanical intermediate is now mostly observed (80% of the occurrences) at ΔL=19.0±0.5 nm (dashed light brown fits), n=78, consistent with the unfolding and extension of the total protein length from the N terminus up to His-46. In the remaining 20% of the cases in which the protein unfolds via a three-state pattern, the protein unfolds via the C terminus, as fingerprinted by the intermediate occurring at ΔL=5.6±1.1 nm, n=18. (d) The histogram corresponding to the increment in contour length elicited after the main unfolding event measured for the Azu_C112A mutant reveals a main unfolding event occurring at ΔL=9.4±1.5 nm, n=62 nm, which matches rather well the broader histogram of the wt-Azu data shown in Fig. 1f (background light grey bars), containing both intermediate events. (e) The normalized histogram corresponding to the unfolding of the Azu_C26A mutant shows well-defined intermediates, occurring at ΔL=6.2±1.0 nm, n=18 and ΔL=19.5±0.7 nm, n=81. Crucially, the N terminus pathway is highly favoured in the absence of the native disulfide bond.

Figure 3. Plastocyanin unfolds through parallel pathways fingerprinted by the rupture of individual Cu–S and Cu–N bonds.

(a) Plastocyanin structure (PDB: 3BQV). (b) Scheme of the (Plastocyanin-I27)₄ polyprotein. (c) Individual constant velocity unfolding trajectory of a single (Plastocyanin-I27)₄ polyprotein, whereby the unfolding of each plastocyanin domain (cyan worm-like chain (WLC) fits) occurs before the unfolding of the I27 markers (purple WLC fits). Inset: Close inspection to each plastocyanin unfolding event reveals that the unfolding process occurs through two distinct mechanical intermediates, which are characterized (d) by an increase in contour length of either ΔL=5.9±1.1 nm, n=66 (green) or ΔL=13.8±1.5 nm, n=82 (brown). In the cases where no intermediate is observed, suggestive of an apoplastocyanin form, unfolding triggers the extension of the complete length of the protein, ΔL=36.8±1.1 nm, n=135 (cyan). (e) Remarkably, the unfolding forces measured for each individual pathway (N terminus, C terminus and two-state unfolding) are very similar (75.3±12 pN, n=82, brown; 65.9±13 pN, n=66, green and 75.8±12 pN, n=135, cyan), respectively. In all cases, protein unfolding occurs at higher forces ∼76 pN than the rupture of (f) the Cu–N_His39 bond (46.0±11 pN, n=82, light blue) or the Cu–S_Cys89 individual bond, occurring at 51.8±13 pN, n=66 (dark blue), as shown in (g).

Figure 4. MD simulations capture the distinct unfolding pathways.

(a) Azurin PDB structure (4AZU). Copper is explicitly shown as a blue sphere, the two sulfur atoms of the disulfide bond Cys3—Cys26 in yellow, the residues trapped after the disulfide bond in white and the rest, in orange. In a steered MD set-up, one of the termini is fixed in the laboratory frame and a constant force is applied to the other one. (b) Unfolding trajectories represented by the time evolution of the protein's end-to-end distance for three representative scenarios: wt-Azu unfolding from the N-terminal up to Gly-45 (brown), from the C-terminal up to Met-121 (green) and unfolding of wt-Azu lacking its disulfide bond from the N-terminal side (light brown). Each simulation was stopped once the end-to-end distance had reached the maximum possible extension at this force, the organometallic bonds being intact. (c) Representative structures following unfolding on the C-terminal side and up to Met-121 (left) and unfolding on the N-terminal side up to Gly-45 (right). (d) Percentage of the population of events falling in each scenario (green: C-terminal unfolding; brown: N-terminal; grey: both simultaneously), for both the oxidized form of wt-Azu (solid bars) and the reduced version lacking its disulfide bond (patterned bars). (e) Unfolding trajectories of wt-plastocyanin (3BQV). In ∼70% of the trajectories (n=7), plastocyanin unfolds through the N terminus (brown). In 20% of the occurrences (n=2), the protein unfolds via the C terminus instead (green), and in the remaining trajectory the protein unfolds from both termini simultaneously (grey). Hence, from a qualitative viewpoint, these in-silico results support the experimental observations.

Figure 5. A generic kinetic partition scheme for the complex mechanical unfolding of wt-azurin and wt-plastocyanin.

On the application of mechanical force, azurin can unfold following multiple pathways. In 20% of the occurrences (40% in the case of plastocyanin), the protein unfolds according to an all-or-none process. By contrast, in 75% of the cases (50% for plastocyanin), azurin unfolds via one mechanically stable intermediate after unfolding from either the N terminus (brown) or the C terminus (green). In the remainder 5% (10%) of the cases, a four-state unfolding pathway involving the simultaneous unfolding of the N and C termini is observed.