Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity.

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)-C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cß-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

Interaction between the anticancer drug Cisplatin and the copper chaperone Atox1 in human melanoma cells.

Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking.

Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.

Role of metal in folding and stability of copper proteins in vitro.

Metal coordination is required for function of many proteins. For biosynthesis of proteins coordinating a metal, the question arises if the metal binds before, during or after folding of the polypeptide. Moreover, when the metal is bound to the protein, how does its coordination affect biophysical properties such as stability and dynamics? Understanding how metals are utilized by proteins in cells on a molecular level requires accurate descriptions of the thermodynamic and kinetic parameters involved in protein-metal complexes. Copper is one of the essential transition metals found in the active sites of many key proteins. To avoid toxicity of free copper ions, living systems have developed elaborate copper-transport systems that involve dedicated proteins that facilitate efficient and specific delivery of copper to target proteins. This review describes in vitro and in silico biophysical work assessing the role of copper in folding and stability of copper-binding proteins. Examples of proteins discussed are: a blue-copper protein (Pseudomonas aeruginosa azurin), members of copper-transport systems (bacterial CopZ, human Atox1 and ATP7B domains) and multi-copper ferroxidases (yeast Fet3p and human ceruloplasmin). The consequences of interactions between copper proteins and platinum-complexes are also discussed. This article is part of a Special Issue entitled: Cell Biology of Metals.

Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1.

Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu-Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.