Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator)

Abstract

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein–protein or protein–DNA interactions.

Visualizing correlated motion with HDBSCAN clustering

“…d), we see the alpha helix once again grouped by itself and the non‐coordinating CYS (gray) separated by the clustering from the majority of the alpha helix. The two orange clusters here contain residues previously seen to exhibit strong anti‐correlation in the presence of zinc—a motion that has been implicated as part of the zinc binding mechanism …”

“…We begin our examples with the more structurally stable of our biological configurations of zinc (Fig. ).…”

“…Additionally, this clustering of the alpha helix residues places each of the zinc binding residues in distinct clusters. The red cluster contains a CYS residue that fails to coordinate with zinc, indicating the clustering has picked out the distinct motion of this portion of the alpha helix.…”

“…Cys . The less stable NEMO configuration is zinc‐unbound and has protonated binding site CYS residues. Using the correlation matrix (Fig.…”

A “Tug of War” maintains a dynamic protein-membrane complex as shown in all-atom simulations of C-Raf RBD-CRD bound to K-Ras4B at an anionic membrane

“…Each zinc finger has one zinc ion surrounded by three Cysteine residues and one Histidine residue. Histidine is in the uncharged HSE form and Cysteine is patched with CYN (Godwin et al, 2017). The ion coordination within the Zinc finger was stable in all simulations.…”