The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM

Stable Cu(II) and Cu(I) Mononuclear Intermediates in the Assembly of the CuA Center of Thermus thermophilus Cytochrome Oxidase

CuA is a dinuclear mixed-valence center located in subunit 2 of the <i>ba</i>₃-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu^1.5–Cu^1.5 system implicates both Cu­(II) and Cu­(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu­(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV–vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu­(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu­(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and that the dominant intermediate along the pathway to mixed-valence is a new green species with λ_max = 460 nm. This intermediate has been isolated in a homogeneous state and shown to be a mononuclear Cu­(II)–(His)­(Cys)₂ species with no observable Cu­(II)–(Met) interaction. Reduction with dithionite generates its Cu­(I) homologue which is again mononuclear but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of the “coupled distortion” model for Cu­(II) thiolates and their relevance to biological metalation reactions of the CuA center