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
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- 作者：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 Valent
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：April 4, 2000
- 年：2000
- 卷：39
- 期：13
- 页码：3611 - 3623
- 全文大小：534K
- 年卷期：v.39,no.13(April 4, 2000)
- ISSN：1520-4995
文摘
Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion throughinteraction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-raycrystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated andfull-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 Å crystal structure ofLYS7 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 yeastCuZnSOD, but it is substantially elongated at one end where the loop regions of the -barrel come togetherto bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicatethat 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 existsin a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward thedimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis forthe specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion intoSOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associateto form a heterodimer via L7D2. The data presented here, however, taken together with previously publishedcrystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model whereina dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer modelover the heterodimer model are enumerated.
-barrel similar to that found in yeastCuZnSOD, but it is substantially elongated at one end where the loop regions of the -barrel come togetherto bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicatethat 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 existsin a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward thedimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis forthe specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion intoSOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associateto form a heterodimer via L7D2. The data presented here, however, taken together with previously publishedcrystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model whereina dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer modelover the heterodimer model are enumerated.