A reaction cycle is proposed for the mechanism of copper−zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following:  (i) The protein backbones (the “SOD rack”) remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 Å from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48−Cu bond distance increases by 0.18 Å. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 Å from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48−Cu distance increases by 0.70 Å, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 Å, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 Å resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domainswapped molecules, which should aid in the design of domainswapped proteins and biomaterials. T hree-dimensional (3D) domain swapping is a mechanism of exchanging one structural domain of a protein monomer with that of the identical domain from a second monomer, resulting in an intertwined oligomer. The swapped domain has nearly identical noncovalent interactions in the oligomer as in the monomer. More than a dozen crystal structures have been determined of dimers and trimers that are 3D domain swapped (1). Domain swapping provides a plausible mechanism for the evolution of functional sites located between the monomeric units of oligomers with well-defined aggregation states (2). 3D domain swapping may also lead to polymerization, and has been suggested as a mechanism of forming protein amyloid fibrils (1, 3). At least two proteins, diphtheria toxin and ribonuclease A, are known to form higher order oligomers (4-6), but the atomic structures of these oligomers are not yet known. These oligomers form under the same conditions as the 3D domain-swapped dimers (DSDs) of known structure, and are presumably also linked by swapping domains.The present paper illustrates through protein design how 3D domain swapping can lead to a DSD or to domain-swapped oligomers, depending on the topology of the monomeric protein.To illustrate this concept, we have prepared domainswapped derivatives of two different, monomeric 3-helix bundles ( Fig. 1 A and B). The bundles (Fig. 1) are variants of the designed 3-␣-helical bundle called coil-Ser, whose design (7-9) was based on the heptad repeat sequence of ␣-helical coiled coils. The 3-␣-helical bundle of coil-Ser has an antiparallel packing arrangement, in which each ␣-helix is made of four heptad repeats containing leucine residues in the a and d heptad positions (Fig. 1C; ref. 10). The crystallographically determined structure of coil-Ser has served as a template for the design of antiparallel three-␣-helix bundles with updown-up topologies...

The three-dimensional structure of the 29-residue designed coiled coil having the amino acid sequence acetyl-E VEALEKK VAALESK VQALEKK VEALEHG-amide has been determined and refined to a crystallographic R-factor of 21.4% for all data from 10-8, to 2.1-8, resolution. This molecule is called coil-V,Ld because it contains valine in the a heptad positions and leucine in the d heptad positions. In the trigonal crystal, three molecules, related by a crystallographic threefold axis, form a parallel three-helix bundle. The bundles are stacked head-to-tail to form a continuous coiled coil along the c-direction of the crystal. The contacts among the three helices within the coiled coil are mainly hydrophobic: four layers of valine residues alternate with four layers of leucine residues to form the core of the bundle. In contrast, mostly hydrophilic contacts mediate the interaction between trimers: here a total of two direct proteinprotein hydrogen bonds are found. Based on the structure, we propose a scheme for designing crystals of peptides containing continuous two-, three-, and four-stranded coiled coils.

The three-dimensional structure of yeast copper-zinc superoxide dismutase (CuZnSOD) has been determined in a new crystal form in space group R32 and refined against X-ray diffraction data using difference Fourier and restrained crystallographic refinement techniques. The unexpected result is that the copper ion has moved approximately 1 angstrom from its position in previously reported CuZnSOD models, the copper-imidazolate bridge is broken, and a roughly trigonal planar ligand geometry characteristic of Cu(I) rather than Cu(II) is revealed. Final R values for the two nearly identical room temperature structures are 18.6% for all 19 149 reflections in the 10.0-1.7 angstrom resolution range and 18. 2% for 17 682 reflections (F > 2 sigma) in the 10.0-1.73 angstrom resolution range. A third structure has been determined using X-ray data collected at -180 degrees C. The final R value for this structure is 19.0% (R(free) = 22.9%) for all 24 356 reflections in the 10.0-1.55 angstrom resolution range. Virtually no change in the positions of the ligands to the zinc center is observed in these models. The origin of the broken bridge and altered Cu-ligand geometry is discussed.