3D structures of the separated C2 and HECT domains of SMURF2 were acquired from the solution structure of C2 2JQZ.pdb27 and the crystal structure of the HECT fragment 1ZVD.pdb28. Initial structure of the HECT–C2 complex was generated by means of the Discovery Studio 4.0 molecular modelling package29, implementing methods ZDOCK30, and RDOCK31 for rigid body protein docking. The docked poses generated by ZDOCK were filtered by a set of C2 residues experiencing the most significant NMR chemical shift in the complex with HECT27. On the next step, RDOCK protocol was used, providing optimisation of docked poses generated by the ZDOCK protocol. RDOCK consists mainly of a two-stage energy minimisation scheme that includes the evaluation of electrostatic and desolvation energies. During the two-stage energy minimisation, RDOCK takes advantage of CHARMM molecular modelling software32 to remove clashes and optimise polar and charge interactions. Finally, the best docking pose was used as input structure for the subsequent MD simulation. Pep3, Pep5, Pep7, and Pep10 designed, synthesised and experimentally estimated in this work as promising inhibitors of SMURF2, were used for the construction of 3D structures of their complexes with HECT by molecular modelling. The best poses of the HECT-peptidyl inhibitor complexes generated by VINA docking algorithm33, implemented in the YASARA structure software34, were used as initial 3D structures for the subsequent MD simulations.

Standard AMBER molecular dynamics software35 procedures were used for the generation of input structural and topological files for all target systems under MD simulations in frames of the FF14SB force field (for protein, peptide substrates, and solvent molecules)36. The generated script files controlling all steps of simulations at 310 K used the 10 Å cut-off. The solute (protein system) was placed in the solvent box with minimal distance of 15 Å to its borders. Solvent water molecules as TIP3P model were filled into the box. The electrostatic charge of the simulated systems was neutralised by addition of Na+ or Cl counter ions. The MD simulated HECT–C2 complex consists of 373 (HECT) and 131 (C2) amino acid residues, 34,219 solvent water molecules, and nine Cl counter anions, constitute 111,219 atoms in total. Both protein fragments of SMURF2, HECT28, and C227, as acquired from the Protein Data Bank, were protected by capping groups at their N- and C-ends by Ac and NMe, respectively. Standard protonation states were used for all ionisable residues: negatively charged Asp, Glu, and positively charged Arg and Lys residues. All His residues where neutral, containing one proton on Nδ except for His 484, 530, and 714 of HECT with one proton on Nε. The Na+ cation was deleted from HECT structure since it was artificially added in the crystallisation procedure28.

The first stage was three sequential steps of energy minimisation:

Minimise only the water, restraining the protein (20,000 cycles).

Let water move (NTP, 300 K), restraining the protein.

Unrestrained minimisation of all system – water and protein (20,000 cycles).

After the initial minimisation, the system was slowly heated in 1 ns from 0 K to the production temperature of 310 K. The Langevin thermostat was used. The SHAKE constraints were used to fix hydrogen atom bond lengths allowing to run with a 2 fs time step. Since in low temperature the calculation of pressure is inaccurate, the response of the barostat can distort the system, so MD simulation was conducted in NVT ensemble. The protein molecule was restrained using harmonic approximation with force constant 10 (kcal/mol)/Å2.

After the system was heated to 310 K, allowing the density of the system to equilibrate, we ran 10 repeated MD restarts each of 500 ps with a time step of 2 fs (SHAKE constraint) under Langevin thermostat and NPT ensemble with pressure of 1 atm. No positional restraints were applied. Random seeds by pseudorandom number generator were used to restart the simulations in repeated segments.

In the previous equilibration stage, the temperature of 310 K and stable density were reached. In this last stage, we ran production dynamics with Langevin thermostat and NPT ensemble under pressure of 1 atm and SHAKE constraint of 2 fs. The total run time is varied for each molecular system depending on its RMSD convergence. In all systems, the MD production simulation started from two 50 ns sequential steps followed by a set of 100 ns steps. Every MD step was restarted from the previous one applying random seeds generator.

By means of CPPTRAJ37,38, implemented in AMBER 16 package, we analysed the RMSD convergence of the MD trajectories, and provided cluster analysis of protein conformations on the equilibrated final fragment of the trajectories in order to identify 3D structure of protein systems corresponding to cluster centroids.

Implemented in AMBER, the MM-PBSA.py method39 was applied for calculation of free energies of binding of peptide inhibitors to HECT in frames of the generalised Born model40. The values of free energies of binding of HECT with peptide inhibitors were calculated on previously generated production MD trajectories. The MD trajectory fragment of 2 ns containing 200 frames centred around the corresponding conformational cluster centroid were used in every simulated HECT-inhibitor complex for the calculation of inhibitor averaged free binding energy. The entropy calculation by performing normal mode analysis was ignored as computationally too expensive and because of a potential source of the results uncertainty.

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