First Principles Calculation of Protein-Protein Dimer Affinities of ALS-Associated SOD1 Mutants

Abstract

Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein-protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid β-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Keywords: amyotrophic lateral sclerosis; dimer dissociation; free energy perturbation; loop entropy; molecular dynamics simulations; protein misfolding (conformational) diseases; protein–protein interactions; superoxide dismutase (Cu–Zn).

FIGURE 1

(A) The three components of the conformational restraints displayed in color, for the SOD1 homodimer. The central barrel backbone is in blue. The backbones of the large flexible loops 4 and 7 are in turquoise. The side chains of the dimer interface residue are in yellow licorice. The structural elements altered in this study are labeled in panel (A) and rendered in red van der Waals spheres. (B) Representation of the local reference frame of WT E,E (SS) used to define chain B position and orientation relative to chain A (see text for a description).

FIGURE 2

Visualization of the stepwise procedure of calculating the binding free energy ΔG _bind. In order to accelerate convergence, restraints are serially applied in the bound state and serially released in the free state. The full thermodynamic process is given in Eq. 11.

FIGURE 3

PMFs for various restraints for each variant. Each row shows the PMF surfaces for the various restraints applied to each given variant, and each column represents a given restraint: loop backbone, barrel backbone, interface sidechain, and inter-monomer separation distance. The PMFs labeled “Bound chain A/Bound chain B” correspond to varying umbrella restraints on the bound states of chain A or B respectively, while the PMFs labeled “free” correspond to varying umbrella restraints on the free state. The separation-distance PMFs are constructed using either the full, the last 75%, or the last 50% of the trajectories in REMD-US, as indicated in the legend.