Structural Insights into the Midnolin Protein: A Molecular Dynamics Approach

The function of the intracellular protein Midnolin was recently elucidated by Gu et al. (2023), who demonstrated its involvement in the degradation of transient, nuclear-acting proteins encoded by immediate-early genes through a non-canonical pathway. The protein degradation triggered by midnolin depends on the proteasome but does not depend on ubiquitination. Structural analyses reveal that this protein is composed of three domains separated by disordered loops: the Catch domain, involved in target and capture of protein substrate; the proteasome-binding domain and the ubiquitin-like domain, which mimics the role of ubiquitin in interacting with the 26S proteasome regulatory subunit. According to structural analyses using modeling and Cryo-EM, the interaction with the target substrate occurs via the formation of an antiparallel beta-sheet with the Catch domain, featuring a steric zipper of phenylalanines and glycines, although a definitive interaction pattern is yet to be established.

In this study, we aim to understand the interaction and specificity mechanisms of the Catch domain with target substrates through classical and accelerated molecular dynamics simulations, proposing the molecular bases of this interaction.

To this end, we conducted molecular dynamics simulations using the ff19SB force field and the OPC water model with 1.500 ns in each system containing the Catch domain and a previously in vitro-validated substrate (10 peptide from EGR1 protein). The initial input structures for both the Midnolin Catch domain and the corresponding ligand were computationally generated utilizing AlphaFold3.We analyzed the conformational stability of the Catch domain in the presence and absence of the ligand by calculating residue RMSD and RMSF.

Furthermore, we evaluated the spontaneous, uninduced docking of the ligands, the energetic landscape (free energy barriers) involved in the process, and the molecular bases through which the substrate interacts with the receptor region. Preliminary results indicate that the ligand establishes consistent and persistent interactions with the surface of the Catch domain. This is mediated by an initial tyrosine-glycine pairing and stabilized by interactions between phenylalanines and glycines from distinct chains.