X-ray Crystallographic and Analytical Ultracentrifugation Analyses of Truncated and Full-Length Yeast Copper Chaperones for SOD (LYS7): A Dimer-Dimer Model of LYS7-SOD Association and Copper Delivery †,‡ Leslie T. Hall, § Raylene J. Sanchez, | Stephen P. Holloway, § Haining Zhu, | Jennifer E. Stine, § Thomas J. Lyons, | Borries Demeler, ⊥ Virgil Schirf, ⊥ Jeffrey C. Hansen, ⊥ Aram M. Nersissian, | Joan Selverstone Valentine, | and P. John Hart* ,§ Center for Biomolecular Structure Analysis and Center for Analytical Ultracentrifugation of Macromolecular Assemblies, Department of Biochemistry, UniVersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl DriVe, San Antonio, Texas 78284-7760, and Department of Chemistry and Biochemistry, UniVersity of California, Los Angeles, California 90095 ReceiVed NoVember 29, 1999; ReVised Manuscript ReceiVed January 24, 2000 ABSTRACT: Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 Å crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key -barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the -barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated. Copper is required for the activation of dioxygen, which is essential for the survival of all living aerobic organisms (1). Paradoxically, the electronic structure of copper that allows its direct interaction with oxygen also renders it quite toxic. Cells therefore tightly regulate the amount of copper allowed into the cytoplasm (2). In addition, cells are armed with a variety of proteins, e.g., the metallothioneins, that scavenge free copper ions. Proteins that use copper ion as a cofactor must somehow acquire it in the face of these scavenging molecules. Knowledge of how this occurs has increased substantially in the last several years with the discovery of a class of molecules termed “copper chaper- ones” (3). The copper chaperones acquire copper either directly or indirectly from the membrane transporter CTR1, protect it from the scavenging molecules (and the cellular environment from it), and deliver and insert it into specific target proteins, thereby activating them (reviewed in ref 4). In eukaryotes, several copper chaperones have been identified, including Cox17, which delivers copper to cyto- chrome c oxidase in the mitochondria (5, 6), Atx1, which delivers copper to vesicular transport ATPases such as Ccc2 (7, 8), and CCS and LYS7, which deliver copper to cytoplasmic human and yeast copper-zinc superoxide dis- mutase, respectively (3). These copper-bearing proteins are highly specific for their targets and cannot functionally substitute for each other in their respective pathways (3). Single-site mutations in CuZnSOD 1 cause the neurode- generative disease familial amyotrophic lateral sclerosis (FALS), the inherited form of Lou Gehrig’s disease (9). A copper-mediated toxic gain of function in FALS mutant CuZnSODs may play a role in motor neuron death (10, 11). † This research was supported in part by grants from the Robert A. Welch Foundation, the University of Texas Health Science Center at San Antonio Research Resources Program for Medical Schools of the Howard Hughes Medical Institute, the University of Texas Health Science Center at San Antonio Competitive Research Enhancement Fund for New Faculty (P.J.H.), and the NIH (GM28222 J.S.V.). ‡ The coordinates have been deposited in the Protein Data Bank (file name 1EJ8). * To whom correspondence should be addressed. E-mail: pjhart@ biochem.uthscsa.edu. Phone: (210) 567-8779. Fax: (210) 567-8778. § Center for Biomolecular Structure Analysis, Department of Bio- chemistry, University of Texas Health Science Center at San Antonio. | University of California. ⊥ Center for Analytical Ultracentrifugation of Macromolecular As- semblies, Department of Biochemistry, University of Texas Health Science Center at San Antonio. 3611 Biochemistry 2000, 39, 3611-3623 10.1021/bi992716g CCC: $19.00 © 2000 American Chemical Society Published on Web 03/10/2000