Our systematic replica exchange DMD simulations indicate a strong coupling of the dimer dissociation and monomer unfolding processes, which has important implications for overall protein stability and aggregation. Dimerization significantly stabilizes the folded monomer, which implies that contacts in the dimer interface contribute to the integrity of the monomer β-barrels. As these contacts are broken, the intra-monomer interactions maintaining the β-barrel formation of each monomer are also broken, causing a simultaneous dissociation and partial unfolding. The unfolding process is then more favorable to complete upon full dissociation.
Similarly, the wild type and mutant unmodified species in general dissociate and unfold more sharply with an increase in temperature than do the modified forms (, Supplementary Figure 1
). This finding implies that the shifting or gain of non-native contacts (Supplementary Figures 13–18
) that occurs in modified species causes a loss of cooperativity in the interface and intra-monomer interactions. This loss of cooperativity allows some contacts to be lost much more frequently than others, whether due to a structural change or the steric interference of the modification molecules. A loss in interaction cooperativity may be manifest in the stabilization of the intermediate state, and also could be the reason behind the decreased potential energy gap between the native and dissociated states (, Supplementary Figures 2–6, Supplementary Table 3
). Interactions between the modifications and their associated monomer may also be responsible for this more drawn out dissociation interaction and loss of cooperativity; both modifications are located near the dimer interface (), and their interactions with the monomers may disrupt or weaken native interface interactions. This effect could possibly be remedied by the introduction of additional interactions in the form of a drug that would bind, bridging the dimer interface and holding it together, as was found by Ray et al38
. In addition, it may be possible to design a drug that would interfere with the modification of SOD1 in the first place, whether by occupation or blocking of the modification site or inhibition of the modification binding itself.
The results above indicate that the post-translational modifications glutathionylation and phosphorylation affect the energetic and structural properties of wild type and mutant SOD1. The effect observed varies both with mutant and with modification. This observation suggests that different types of modification have varied effects on the stability of dimer of the various genetic mutations, and for different reasons. For example, A4V dimer seems to be largely stabilized by modification, while the wild type dimer is destabilized by glutathionylation but appears to be “rescued” from this effect by phosphorylation. With the exceptions of A4V and I112T, glutathionylation in particular has a dramatic effect in decreasing the potential energy gap between the native-like state and the dissociated state. We infer from this that the presence of glutathione, a marker of oxidative stress in the cell39
, would be detrimental in most types of familial ALS. This finding corroborates with reports that exercise40
and electrical stimulation41
of ALS model animals results in a more rapid and severe disease progression, since both of these would produce increased oxidative stress in cells and hence increased levels of glutathionylated protein42
. An environmental factor such as oxidative stress could also help to explain the differences in disease progression between the various ALS-causative mutants.
Our simulation results indicate that post-translational modifications in wild type and mutant A4V SOD1 result into a net loss in the total number of interface contacts, and thus, a reduced binding energy. This result is consistent with the experimental observation of reduced dimer dissociation constant in modified wild type and A4V mutant (Redler, R. L., K. C. Wilcox, E. A. Proctor, L. Fee, M. Caplow, and N. V. Dokholyan (2011) submitted). Our simulations of the I112T mutant also suggest that the modification induces a shift in the dimer interface, but no significant change in the total number of contacts, in contrast to the wild type and A4V mutant. This result suggests that modified I112T has a similar dimer dissociation constant to the unmodified species. Indeed, Redler et al. have shown that the equilibrium constant of I112T dimer dissociation is not affected by glutathionylation.
In this work, we study homo-modified species of wild type and mutant SOD1. However, it is possible that in vivo
SOD1 may exhibit significant hetero-modified populations in addition to homo-modified species. Because of the means of experimental characterization of post-translational modifications from erythrocytes (mass spectrometry)29
(Redler, R. L., K. C. Wilcox, E. A. Proctor, L. Fee, M. Caplow, and N. V. Dokholyan (2011) submitted), dimers are necessarily dissociated before measurement, and so it is impossible to determine whether dimers are hetero- or homo-modified in vivo
using this method. The molecular mechanism of glutathionylation, as well as the kinase responsible for phosphorylation of SOD1, is still unknown, so it is unclear whether modification of the individual monomers is a cooperative or an independent process. However, even the presence of only one modification molecule near the dimer interface disrupts or changes monomer-monomer contacts and induces many of the same effects that we observe in homo-modified species.
On a further note, because glutathionylation decreases the potential energy gap between the native-like and dissociated states in wild type SOD1, and glutathionylation of SOD1 is present even in healthy individuals29
, it is possible that glutathionylation caused by oxidative stress to motor neurons could be a factor in sporadic ALS. The late onset of ALS suggests that an environmental trigger could exist for both familial and sporadic cases. This possibility fits with the increased occurrence of sporadic ALS in athletes43
as compared to the general population; both of these groups experience more extreme and frequent oxidative stress than does the average individual. Such environmental factors could possibly be counteracted with a drug or lifestyle decisions. Further investigation into the mechanism of post-translational modification in SOD1 could illuminate preventative measures against the observed increased dissociation and unfolding, and hence inhibit aggregation and disease.