Structural Discovery of Small Molecule Binding Sites in Cu−Zn Human Superoxide Dismutase Familial Amyotrophic Lateral Sclerosis Mutants Provides Insights for Lead Optimization

Dominant inheritance of point mutations in CuZn superoxide dismutase (SOD1) is the best characterized subset of familial amyotrophic lateral sclerosis (FALS) and accounts for some 20% of the known familial cases. We report the discovery and visualization via cocrystallography of two ligand-binding pockets in human SOD1 and its pathogenic mutants that have opened up the real possibility of undertaking lead compound discovery using a fragment-based approach for therapeutic purposes for SOD1 associated motor neuron disease

Data Collection and Refinement Statistics.

<p>R_merge = Σ|I_hkl-hkl>|/ΣI_hkl.</p><p>R_cryst = Σ|F_obs-F_calc|/ΣF_obs.</p><p>R_free : R-factor using a subset of 5% of random reflections excluded from refinement.</p><p>ML ESU: Maximum likelihood estimated standard uncertainty in REFMAC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044811#pone.0044811-Murshudov1" target="_blank">[49]</a>.</p><p>Values in parentheses refer to the outermost resolution shell.</p

The active sites of reduced (A) and oxidised (B) SOD in the presence of carbonate.

<p>The copper and zinc ions are shown as cyan and orange spheres, respectively. In the oxidised enzyme, the carbonate anion is located ∼5 Å from the Cu(II) atom, forming H-bonds with the NE and NH2 atoms of Arg143. A well-ordered network of water molecules occupies the active site channel (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044811#pone-0044811-g003" target="_blank">figure 3</a>), with W0 coordinated to the copper atom of the oxidised enzyme. In the reduced enzyme, the carbonate anion is directly coordinated to the Cu(I) atom, displacing W0. Water molecules W1 and W2 are conserved in both structures and play a significant role during superoxide binding and catalysis (see main text). The 2Fo-Fc electron density maps are shown contoured at 1.2σ.</p

The entry and location of carbonate anions at the active site.

<p>Upper panel: Superposition of the metal binding regions of the crystal structure subunit A (red), which contains Cu(I), with subunit C (blue), which contains Cu(II). Upon reduction of the copper, the carbonate anion moves into the cavity, the copper atom moves toward carbonate and the His63 is rotated away from the copper towards carbonate. These changes are facilitated by the opening up (by ∼1 Å) of the active site cavity by a reorientation of the Thr137 main chain and neighbouring residues making up the short 3/10-helix region of the electrostatic loop. The side of the cavity formed by Arg143 and adjacent residues of the electrostatic loop are relatively unperturbed. Lower panel: The electrostatic potential of SOD plotted on the solvent-accessible surface, from +10kT/e (blue) to −10kT/e (red). The positive potential at the active site channel leading towards the copper atom is shown with both positions for the carbonate anion superimposed. The surface is displayed transparently to reveal the Arg143 and Thr137 electrostatic loop residues at the mouth of the channel, the copper atom at the base of the channel and the zinc atom.</p

Stereo view of superoxide modeled in the SOD active site with carbonate.

<p>A 10 Å sphere about the copper atom (cyan sphere) is shown that captures the zinc (orange sphere) binding site and conserved water molecules (red spheres). The H-bonding networks around the carbonate anion (black stick) and the proposed location of the superoxide (purple stick) are indicated by dashed red lines. In the oxidised enzyme (A), the superoxide molecule is shown in position for inner-sphere electron transfer, where it is coordinated directly to Cu(II), displacing water W0 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044811#pone-0044811-g001" target="_blank">figure 1</a> for labeling scheme) and H-bonding with W1. In the reduced enzyme structure (B) carbonate is bound in place of W0 and blocks direct access of superoxide to the Cu(I) atom. The superoxide molecule is shown superimposed at the location normally occupied by conserved water W1, where it is in position to participate in outer-sphere electron transfer from the Cu(I) and simultaneous proton transfer from carbonate.</p

Conserved and ordered solvent molecules from the surface of the enzyme to the active site.

<p>Twenty structures of human, bovine and yeast SOD1 are superimposed. The positions of W0 (site of the first Cu(II)-bound superoxide), W1 (which we propose here is the binding site of the second ‘outer sphere’ superoxide molecule in the Cu(I) enzyme, see main text) and W2 (part of the proton channel) are indicated. The locations of azide (PDB ids: 1sxz, cyan and 1yaz, pale orange), thiocyanate (PDB id: 1sxs, orange) and carbonate anions (shown in red and blue) are also shown in close up in the lower panel: the azide and thiocyanate anions are oriented so that their terminal atoms occupy positions normally taken up by water molecules W0 and W1, while carbonate is found at position W0 in the Cu(I) enzyme (red). The superimposed structures are taken from the RSCB Protein Databank. Human SOD1∶11zv, 1n18, 1n19, 1ptz, 1pu0, 1uxm, 1hl5, 1sos. Bovine SOD1∶1sxs, 1sxz, 1e90, 1q0e, 1cb4, 1cbj. Yeast SOD1∶1yaz, 2jcw, 1yso, 1jcv, 1sdy.</p

A Tetranuclear Cu(I) Cluster in the Metallochaperone Protein CopZ

Copper trafficking proteins and copper-sensitive regulators are often found to be able to bind multiple Cu(I) ions in the form of Cu(I) clusters. We have determined the high-resolution X-ray crystal structure of an Atx1-like copper chaperone protein from Bacillus subtilis containing a novel tetranuclear Cu(I) cluster. The identities and oxidation states of the cluster ions were established unambiguously by refinement of X-ray energy-dependent anomalous scattering factors. The [Cu4(S-Cys)4(N-His)2] cluster geometry provides new structural insights into not only the binding of multiple cuprous ions by metallochaperones but also protein-associated tetranuclear Cu(I) clusters, including those found in eukaryotic copper-responsive transcription factors

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