  Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free
radical defense enzyme whose misfolding and aggregation play a potential key
role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative
disease. Over 150 mutations in SOD1 have been identified with a familial form
of the disease, but it is presently not clear what unifying features, if any,
these mutants share to make them pathogenic. Here, we develop several new
computational assays for probing the thermo-mechanical properties of both
ALS-associated and rationally-designed SOD1 variants. Allosteric interaction
free energies between residues and metals are calculated, and a series of
atomic force microscopy experiments are simulated with variable tether
positions, to quantify mechanical rigidity "fingerprints" for SOD1 variants.
Mechanical fingerprinting studies of a series of C-terminally truncated
mutants, along with an analysis of equilibrium dynamic fluctuations while
varying native constraints, potential energy change upon mutation,
frustratometer analysis, and analysis of the coupling between local frustration
and metal binding interactions for a glycine scan of 90 residues together
reveal that the apo protein is internally frustrated, that these internal
stresses are partially relieved by mutation but at the expense of metal-binding
affinity, and that the frustration of a residue is directly related to its role
in binding metals. This evidence points to apo SOD1 as a strained intermediate
with "self-allostery" for high metal-binding affinity. Thus, the prerequisites
for the function of SOD1 as an antioxidant compete with apo state
thermo-mechanical stability, increasing the susceptibility of the protein to
misfold in the apo state.
