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.

Ensemble Generation for Linear and Cyclic Peptides Using a Reservoir Replica Exchange Molecular Dynamics Implementation in GROMACS

“…US was performed serially, i.e., the final coordinate of a given US window served as the initial coordinate of the next US window. Thus, each successive window benefits at least partially from a longer equilibration time . The simulation took 5 ns for each of the 55 windows generated during serial window construction.…”

“…Thus, each successive window benefits at least partially from a longer equilibration time. 96 The simulation took 5 ns for each of the 55 windows generated during serial window construction. Subsequently, each window was extended for another 35 ns (Figure 3a).…”

Investigating the hepatoprotective potentiality of marine-derived steroids as promising inhibitors of liver fibrosis

“…SOD is a homodimer that converts toxic oxygen radicals to less harmful species. 63,64 The catalytic pocket is defined by Cu ion and residues of His63 and Arg143, 63 Fig. 3B.…”

Exploring the Folding Mechanism of Dimeric Superoxide Dismutase

“…The first peak of the barrier to start the unbinding the apo dimer at 25 °C is about 9 ± 0.5 kcal/mol, and at the maximum distance that the centers of mass are separated in the simulation (42 Å), it is about 10 kcal/mol, a value not very different from an experimental data of 11 ± 0.4 kcal/mol at 23 °C measured by Isothermal Titration Calorimetry . A recent study discusses the differences between the free energy of binding obtained from simulations and experimental data …”