2.2.1. Generation of preliminary 3D structures of molecular systems: HECT–C2 and HECT–peptide inhibitor complexes
This protocol is extracted from research article:
Development and characterisation of SMURF2-targeting modifiers
J Enzyme Inhib Med Chem, Jan 12, 2021; DOI: 10.1080/14756366.2020.1871337

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.

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