Cu,Zn-Superoxide Dismutase without Zn Is Folded but Catalytically Inactive

Abstract

Amyotrophic Lateral Sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding, and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding, but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using Quantum Mechanics/Discrete Molecular Dynamics, we showed that Zn-less wild type SOD1 and its D124N mutant that does not bind Zn both have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I) – Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the wild type SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less wild type SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

Computational Treatment of Metalloproteins

“…A combined computational and experimental study addressed the effect of Zn removal on the protein structure, electronic structure of the Cu site, and overall catalytic function of SOD. 47 The results show that Zn plays a structural role in SOD1 and directly influences the catalysis, enabling proper coordination and reduction potential of the Cu site. Removal of Zn causes the elimination of the catalytic activity of SOD1 even without protein unfolding and aggregation.…”

“…46 For the equilibrated QM/DMD structures, one can apply most rigorous QM methods to obtain such sensitive properties as changes in the reduction potentials of the metal or barriers of catalyzed reactions. 42 Naturally, since sampling is done on the entire protein while metal coordination is purely in the QM management, events such as ligand attachment 44 or detachment 47 are easily captured. This is often important in mechanistic studies.…”

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

“…One part of the metalloprotein design problem is structural: predictions of metal coordination, protein backbone structures, and substrate binding require considering the induced fit effect. For the structural equilibration this study employs our QM/DMD 48 50,51,[55][56][57][58][59][60] Additionally, the speed of QM/DMD is due to the construction of the QM-DMD boundary (see below). QM/DMD allows us to address the vast timescales that metalloenzymes operate under, from the chemical transformation to the large-scale motions that occur on the nano-, milli-and even second timescale.…”

Use of QM/DMD as a Multiscale Approach to Modeling Metalloenzymes

“…QM/DMD has been tested and shown to perform well for such tasks as recapitulation of natural protein structures , recovery of these structures back to the equilibrium after moderate distortions such as temporary removal and reinstallation of the metal , prediction of structural changes upon mutagenesis , flexible docking of substrates to metalloproteins that involve motion of large protein parts such as loops , predictions of protein structure after replacement of the metal or change of its oxidation state and accompanying changes in the coordination sphere geometry and number of ligands (Nedd, Redler, Proctor, & Dokholyan, 2014;Valdez, Gallup, & Alexandrova, 2014), and predictions of protein rearrangements after removal of the metal in cases where the protein does not completely unfold, or the use of a short sequence within 20-30 amino acids (Nedd et al, 2014). Structures produced by QM/DMD are in good agreement with quality X-ray structures, if available .…”