title: Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein.
abstract: The thermodynamics of zinc binding to metal-free (apo) human and bovine copper-zinc superoxide dismutases (SOD1) were measured using isothermal titration calorimetry. The apparent thermodynamics of zinc binding to the apoproteins were favorable (Ka > 108 M-1), with an observed stoichiometry of one zinc per homodimer. The change in heat capacity for the one-zinc binding event was large and negative (approximately -650 cal mol-1 K-1), suggestive of significant structural changes to the protein upon zinc binding. We further characterized the one-zinc derivative by circular dichroism and determined that this derivative had nearly the same secondary structure as the two-zinc derivative and that both are structurally distinct from the metal-free protein. In addition, we monitored the effect of zinc binding on hydrogen-deuterium exchange and accessibility of histidyl residues to modification by diethyl pyrocarbonate and observed that more than 50% protection was afforded by the binding of one zinc in both assays. Differential scanning calorimetry on the human SOD1 zinc derivatives also showed increased thermostability of the protein due to zinc binding. Further, the melting transitions observed for the one-zinc derivative closely resembled those of the two-zinc derivative. Finally, we observed that the quaternary structure of the protein is stabilized upon binding of one and two zinc ions in analytical ultracentrifugation experiments. Combined, these results suggest communication between the two monomers of SOD1 such that the binding of one zinc ion per homodimer has a more profound effect on the homodimeric protein structure than the binding of subsequent metal ions. The relevance of these findings to amyotrophic lateral sclerosis is discussed.
authors: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, CA 90095, USA.
authors: Soshanna Zittin
authors: SZ
authors: Potter
authors: Haining
authors: H
authors: Zhu
authors: Bryan Francis
authors: BF
authors: Shaw
authors: Jorge A
authors: JA
authors: Rodriguez
authors: Peter A
authors: PA
authors: Doucette
authors: Se Hui
authors: SH
authors: Sohn
authors: Armando
authors: A
authors: Durazo
authors: Kym F
authors: KF
authors: Faull
authors: Edith Butler
authors: EB
authors: Gralla
authors: Aram M
authors: AM
authors: Nersissian
authors: Joan Selverstone
authors: JS
authors: Valentine
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D002412:Cations / Q000737:chemistry*; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry*
chemical_list: D001059:Apoproteins; D002412:Cations; D021122:Protein Subunits; D013482:Superoxide Dismutase; D015032:Zinc