Molecular dynamics simulation reveals insights into the mechanism of unfolding by the A130T/V mutations within the MID1 zinc-binding Bbox1 domain

Abstract

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.

Fig 1. Structural properties of the MID1 Bbox1 domain.

(A) The ensemble of 13 structures generated from NMR-derived restraints (PDB code: 2FFW). (B) Magnitude of the fluctuation represented as eigenvectors of the Bbox1 ensemble of structures. The conformational fluctuations indicate both magnitude and directions (arrows) and are derived from PCA analysis. (C) The fluctuations of the average NMR structure are shown as a function of residue number. (D) Zoomed-in snapshot of the small cavity near residue A130. The blue dots represent the solvent accessible surface.

Fig 2. Effects of mutating of residue A130.

(A) Homology modeling of Bbox1 structures highlighting the position and packing of side chain of A130 (i) compared to those of the T130 (ii) and V130 (iii). (B) Backbone RMSDs for WT (blue), A130T (red), and A130V (orange) during the MD simulations.

Fig 3. Structural analysis of the MD simulation results.

(A) Backbone RMSFs for WT (blue), A130T (red), and A130V (orange) during the MD simulations. (B) Time evolution of the secondary structural elements, based on DSSP classification, of the wild type and mutant proteins. (C) The angle formed by the Cα atoms of D129, A130, and V131. (D) The angle fluctuations for WT (blue), A130T (red), and A130V (orange) during the MD simulations with the corresponding histogram to the side.