Molecular Mechanisms of Copper Delivery by Copper Chaperone for Sod1 (Ccs1)

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Molecular Mechanisms of Copper Delivery by Copper Chaperone for Sod1 (Ccs1)

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Title: Molecular Mechanisms of Copper Delivery by Copper Chaperone for Sod1 (Ccs1)
Author(s):
Boyd, Stefanie Dionne
Advisor: Winkler , Duane D
Date Created: 2017-05
Format: Dissertation
Keywords: Superoxide dismutase
Molecular chaperones
Macromolecules
Copper ions
Abstract: Metallo-chaperones are a diverse family of trafficking molecules that deliver metal ions to macromolecular targets located within multiple cellular compartments. These “helper-proteins” have evolved distinct characteristics to facilitate selective metal ion binding, target recognition, and cargo transfer. One such recipient is Cu,Zn Superoxide Dismutase (Sod1), an antioxidant metallo-enzyme that scavenges the free radicals produced by cellular respiration. The copper chaperone for Sod1 (Ccs1) is known to provide a copper ion to Sod1 while also completing activation by catalyzing formation of a critical intramolecular disulfide bond. Here, we outline a structure-based approach used to define in detail the molecular mechanisms involved in Ccs1-mediated activation of Sod1. A new crystal structure of Ccs1 in complex with an immature Sod1 monomer suggests that binding of Ccs1 induces significant conformational changes to Sod1 that exposes a previously unobserved copper ion “entry site” or “drop-off-point” for Ccs1. Quantitative thermodynamic binding assays and Sod1 activation experiments performed in vitro support a “pivot and release” model for Ccs1-mediated delivery of copper to Sod1 during copper-limiting conditions. Interestingly, our results also suggest that under copper-replete conditions the main job of Ccs1 may not directly involve copper-ion hand-off to Sod1, but to simply expose the “entry site” for copper delivery by another copper-bound small molecule, likely glutathione. X-ray absorption spectroscopy (XAS) and cell-based biochemical assays provide evidence that Ccs1 promotes copper ion coordination at an “entry site” away from the Sod1 active site; redox turnover of superoxide at this site leads to a sulfenic acid intermediate that resolves into the stable Sod1 disulfide bond and release of the copper ion into the Sod1 active site. In addition, experiments measuring the ability of Sod1 to bind and retain zinc ions indicate that interaction with Ccs1 facilitates the binding of this metal to Sod1 in the absence of copper or the intramolecular disulfide bond. Ccs1 may actually perform dual chaperoning roles for Sod1, acting as both a copper and molecular chaperone.
Degree Name: PHD
Degree Level: Doctoral
Persistent Link: http://hdl.handle.net/10735.1/5391
Type : text
Degree Program: Biology - Molecular and Cell Biology

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