In view of performing molecular dynamic simulations, we first generated structural models of wt and IPKI MDs prolonged to cover the entire MD (amino acids 304 to 375) and hence to include the dynamic domain (D) to be able to monitor over time the possible structural changes occurring within this region. In addition, the starting model of wt P MD was chosen to be symmetric, i.e., to have a kink on each of the four chains. The rationale for this choice resides in the fact that the asymmetry of the 3ZDO structure likely arises from crystal packing artifacts (19). To generate the prolonged wt and IPKI MD models for molecular dynamic simulations, the structures of wt P MD (PDB code 4C5Q) and IPKI variant (PDB code 6HTL) were used as starting models complemented by the C terminus of the 3ZDO structure (residues 357 to 370). The alignment of the query and template sequences was first performed by means of the MUSCLE web server (84) with the Pearson output file format. The resulting target alignment file was used as input to generate a 3D structural model of MD using MODELLER (85). The models of MD variants bearing a substitution at position 315 were built in the same way by means of the MODELLER software as described above.

Molecular dynamics simulations were carried out by means of the ACEMD software (86) with the AMBER99SB-ILDN force field (87) and with the TIP3P water model (88). The systems were minimized and equilibrated under constant pressure (1 atm) and temperature (300 K) [constant number of particles (N), pressure (P) and temperature (T), NPT] conditions using a time step of 4 fs [owing to the use of the hydrogen mass repartitioning scheme (89) implemented in ACEMD], a nonbonded cutoff of 9 Å, and particle-mesh Ewald long-range electrostatics with a grid of 70 × 74 × 138 with spacing of 1 Å. The systems were equilibrated first using 2000 steps of steepest-descent minimization, followed by running 0.12 ns of the isothermal NVT [constant number of particles (N), volume (V), and temperature (T)] ensemble, using a Langevin thermostat set at 300 K, followed by 2.8 ns of the isothermal-isobaric NPT ensemble using a Langevin thermostat at the same temperature and the Berendsen barostat of ACEMD. During minimization and equilibration, the heavy atoms were restrained spatially using a spring constant (1 kcal mol−1 Å−2). Then, a production run of 1 μs in the NVT ensemble was performed. The processing of the different trajectories and the secondary structure timeline analysis were performed by using VMD (90) and the GROMACS dssp utility (91). Normal mode analysis was performed by means of the NOLB software (92) that is able to generate structures with a fewer number of geometric distortions compared to the linear approach. To represent the amplitude of the normal modes, we generated 100 frames for wt P MD and for the IPKI, V315I, V315L, and V315N variants. The amplitude of the different curvatures was then analyzed by means of the Bendix module (93) of VMD, while the isosurface curvature map was displayed within MATLAB (MathWorks Inc).

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