Molecular dynamics of a thermostable multicopper oxidase from Thermus thermophilus HB27: structural differences between the apo and holo forms

Abstract

Molecular dynamic (MD) simulations have been performed on Tth-MCO, a hyperthermophilic multicopper oxidase from thermus thermophilus HB27, in the apo as well as the holo form, with the aim of exploring the structural dynamic properties common to the two conformational states. According to structural comparison between this enzyme and other MCOs, the substrate in process to electron transfer in an outer-sphere event seems to transiently occupy a shallow and overall hydrophobic cavity near the Cu type 1 (T1Cu). The linker connecting the β-strands 21 and 24 of the second domain (loop (β21-β24)(D2)) has the same conformation in both states, forming a flexible lid at the entrance of the electron-transfer cavity. Loop (β21-β24)(D2) has been tentatively assigned a role occluding the access to the electron-transfer site. The dynamic of the loop (β21-β24)(D2) has been investigated by MD simulation, and results show that the structures of both species have the same secondary and tertiary structure during almost all the MD simulations. In the simulation, loop (β21-β24)(D2) of the holo form undergoes a higher mobility than in the apo form. In fact, loop (β21-β24)(D2) of the holo form experiences a conformational change which enables exposure to the electron-transfer site (open conformation), while in the apo form the opposite effect takes place (closed conformation). To confirm the hypothesis that the open conformation might facilitate the transient electron-donor molecule occupation of the site, the simulation was extended another 40 ns with the electron-donor molecule docked into the protein cavity. Upon electron-donor molecule stabilization, loops near the cavity reduce their mobility. These findings show that coordination between the copper and the protein might play an important role in the general mobility of the enzyme, and that the open conformation seems to be required for the electron transfer process to T1Cu.

Figure 1. Structural topology of holo-Tth-MCO and superposition with others MCÓs.

A) Multinuclear metal site of the holo-Tth-MCO. B) Structural depiction of the three cupredoxin domains of holo-Tth-MCO, (domain 1, green), (domain 2, light blue) and (domain 3, yellow), the loop (β21–β24)_D2 is in deep blue cartoons. C) Ribbon superposition of the holo forms of Tth-MCO (green), CueO (red, PDB entry 1N68) and CotA (blue, PDB entry 1UVW), the main differences among them are represented in cartoon structure. Mononuclear copper center (T1Cu) is shown in blue and trinuclear copper cluster (TNC) is shown in orange (T3Cu) and red (T2Cu) Van der Waals representation.

Figure 2. Root mean square deviation (RMSD) from the crystallographic structures of the C^α atoms as a function of simulation time for apo-Tth-MCO form (black line), holo-Tth-MCO (red line) and holo-Tth-MCO without loop (β21–β24)_D2 (blue line).

Figure 3. Structural evidences of the average conformations accessible in the 38 ns MD simulation for the holo-Tth-MCO (snapshots A–C) and apo-Tth-MCO forms (snapshots D–E).

The different secondary structure elements are represented in light blue cartoons. The protein regions involved in the open-closure of the electron-transfer site are in deep blue cartoons: Loop (β21–β24)_D2 and α4-helix_D2 for snapshots A-B and D, loop (β21–β24)_D2 and (187-α4-201)_D2 for snapshot C, loop (β21–β24)_D2, loop (β25–β26)_D3 and loop (β28–β29)_D3 for snapshot E.

Figure 4. Root mean square deviations (RMSD) from the starting structure of the C^α atoms, as a function of the residue number, for apo-Tth-MCO (black) and holo-Tth-MCO (red) for the last 18 ns of a 38 ns-long simulation.

Deviations are averaged over C^α fragments with a homogeneous secondary structure. Error bars represent the standard deviation. Secondary structure elements are shown at top: β-sheet (black) and α-helix (grey).

Figure 5. Eigenvalues as a function of the first 15 eigenvectors, in the time interval 20–38 ns, for apo-Tth-MCO (blue solid square) and holo-Tth-MCO (red solid circles).

Figure 6. Fluctuations of the C^α atoms in the first (A) and the second (B) eigenvector for the time interval 20–38 ns, as a function of the residue number, for apo-Tth-MCO form (square black symbol) and holo-Tth-MCO form (circle red symbol).

Figure 7. Cross-correlation motions (DCCM) for apo-Tth-MCO form (panel A) and holo-Tth-MCO (panel B).

DCCM larger than 0.5 nm are shown on the upper triangle and all values on the lower triangle. Vector products representing the maxim extent of correlated motion (nm) for each C^α pair are plotted. The color scale indicates the degree of correlation: red, positively correlated; blue, negatively correlated; white, uncorrelated.

Figure 8. RMSF analysis of the C^α atoms of TNC’s residues (A) and the second sphere carboxylate residues (B), for apo-Tth-MCO (black) and holo-Tth-MCO (red) for the last 18 ns of a 38 ns-long simulation.

Figure 9. Average conformations of ABTS inside the electron-transfer complex formed with holo-Tth-MCO electron-transfer cavity during the last 25 ns of MD simulation. Residues in close proximity (<0.4 nm) to the ABTS (orange stick models) are represented as green stick models and coppers are shown in Van der Waals representation.

B) The non-bonded short-range Coulomb (black line) and Lennard–Jones (red line) interaction between holo-Tth-MCO and ABTS as a function of time.