Dataset Information

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity.

ABSTRACT: The connecting peptide (C-peptide) is a hormone with promising health benefits in ameliorating diabetes-related complications, yet mechanisms remain elusive. Emerging studies point to a possible dependence of peptide activity on bioavailable metals, particularly Cu(II) and Zn(II). However, little is known about the chemical nature of the interactions, hindering advances in its therapeutic applications. This work uncovers the Cu(II)-binding site in C-peptide that may be key to understanding its metal-dependent function. A combination of spectroscopic studies reveal that Cu(II) and Zn(II) bind to C-peptide at specific residues in the N-terminal region of the peptide and that Cu(II) is able to displace Zn(II) for C-peptide binding. The data point to a Cu(II)-binding site consisting of 1N3O square-planar coordination that is entropically driven. Furthermore, the entire random coil peptide sequence is needed for specific metal binding as mutations and truncations reshuffle the coordinating residues. These results expand our understanding of how metals influence hormone activity and facilitate the discovery and validation of both new and established paradigms in peptide biology.

SUBMITTER: Stevenson MJ

PROVIDER: S-EPMC8023225 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Publications

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity.

Stevenson Michael J MJ Janisse Samuel E SE Tao Lizhi L Neil Ryan L RL Pham Quang D QD Britt R David RD Heffern Marie C MC

Inorganic chemistry 20200608 13

The connecting peptide (C-peptide) is a hormone with promising health benefits in ameliorating diabetes-related complications, yet mechanisms remain elusive. Emerging studies point to a possible dependence of peptide activity on bioavailable metals, particularly Cu(II) and Zn(II). However, little is known about the chemical nature of the interactions, hindering advances in its therapeutic applications. This work uncovers the Cu(II)-binding site in C-peptide that may be key to understanding its m ...[more]

PMID: 32510934

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Project description:UnlabelledUptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive (64)Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter.ImportanceCu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer.

Project description:Alzheimer's disease (AD) is the most common form of dementia worldwide. AD brains are characterized by the accumulation of amyloid-β peptides (Aβ) that bind Cu2+ and have been associated with several neurotoxic mechanisms. Although the use of copper chelators to prevent the formation of Cu2+-Aβ complexes has been proposed as a therapeutic strategy, recent studies show that copper is an important neuromodulator that is essential for a neuroprotective mechanism mediated by Cu2+ binding to the cellular prion protein (PrPC). Therefore, in addition to metal selectivity and blood-brain barrier (BBB) permeability, an emerging challenge for copper chelators is to prevent the formation of neurotoxic Cu2+-Aβ species without perturbing the neuroprotective Cu2+-PrPC interaction. Previously, we reported the design of a tetrapeptide (TP) that withdraws Cu2+ from Aβ(1-16) and impacts the Cu2+-induced aggregation of Aβ(1-40). In this study, we improved the drug-like properties of TP in a BBB model, evaluated the metal selectivity of the optimized peptide (TP*), and tested its effect on Cu2+ coordination to PrPC and proteins involved in copper trafficking, such as copper transporter 1 and albumin. Our results show that changing the stereochemistry of the first residue prevents TP degradation in the BBB model and coadministration of TP with a peptide that increases BBB permeability allows its passage through the BBB model. TP* is highly selective toward Cu2+ in the presence of Zn2+ ions, transfers Cu2+ to copper-trafficking proteins, and forms a ternary TP*-Cu2+-PrP species that does not perturb the physiological conformation of PrP and displays only a minor impact in the neuroprotective Cu2+-dependent interaction of PrPC with the N-methyl-d-aspartate receptor. Overall, these results show that TP* displays desirable features for a copper chelator with therapeutic potential against AD. Moreover, this is the first study that explores the effect of a Cu2+ chelator with therapeutic potential for AD on Cu2+ coordination to PrPC (an emerging key player in AD pathology), integrating recent knowledge about metalloproteins involved in AD with the design of copper chelators against AD.

Dataset Information

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity.

Publications

Elucidation of a Copper Binding Site in Proinsulin C-peptide and Its Implications for Metal-Modulated Activity.

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