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Structure and dynamics of copper-free SOD: The protein before binding copper

The Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, 50019, Sesto Fiorentino, Florence, Italy

Reprint requests to: Professor Ivano Bertini, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; e-mail: bertini{at}cerm.unifi.it; fax: 39-055-4574271.

The solution structure of the copper-free state of a monomeric form of superoxide dismutase (153 amino acids) was determined through ¹³C and ¹⁵N labeling. The protein contained two mutations at the native subunit–subunit interface (F50E and G51E) to obtain a soluble monomeric species and a mutation in the active site channel (E133Q). About 93% of carbon atoms, 95% of nitrogen atoms, and 96% of the protons were assigned. A total of 2467 meaningful NOEs and 170 dihedral angles provided a family of 35 conformers with RMSD values of 0.76 ± 0.09 Å for the backbone and 1.22 ± 0.13 Å for all heavy atoms. The secondary structure elements, connected by loops, produce the typical superoxide dismutase Greek key fold, formed by an eight-stranded ß-barrel. The comparison with the copper-bound monomeric and dimeric structures shows that the metal ligands have a conformation very close to that of the copper-bound forms. This feature indicates that the copper-binding site is preorganized and well ordered also in the absence of the copper ion. The active-site channel shows a sizable increase in width, achieving a suitable conformation to receive the copper ion. The histidines ring NH resonances that bind the copper ion and the region around the active-site channel experience, as found from ¹⁵N relaxation studies, conformational exchange processes. The increased width of the channel and the higher mobility of the histidine rings of the copper site in the copper-free form with respect to the holoprotein is discussed in terms of the process of copper insertion.

Keywords: Copper-free superoxide dismutase; solution structure; NMR; Protein mobility

Abbreviations: CCS, copper chaperone for SOD • SOD, superoxide dismutase • M2SOD, Phe50Glu, Gly51Glu superoxide dismutase • M2E133QSOD, Phe50Glu, Gly51Glu, Glu133Gln superoxide dismutase • M4SOD, Phe50Glu,Gly51Glu, Val148Lys, Ile151Lys superoxide dismutase • EZnM2E133QSOD, empty, zinc M2E133QSOD • TPPI, time-proportional phase incrementation • NOESY, nuclear Overhauser effect spectroscopy • HSQC, heteronuclear single quantum coherence • TOCSY, total correlation spectroscopy • CPMG, Carr-Purcell-Meiboom-Gill • WATERGATE, water suppression by gradient-tailored excitation • NOE, Nuclear Overhauser effect • CSI, chemical shift index • REM, restrained energy minimization • R1, longitudinal relaxation rate • R2, transverse relaxation rate • RMSD, root-mean-square deviation

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