Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein – a lesson in quantification of metal binding to proteinsvialigand competition

Abstract

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Stabilization of Aliphatic Phosphines by Auxiliary Phosphine Sulfides Offers Zeptomolar Affinity and Unprecedented Selectivity for Probing Biological CuI

“…While bathocuproine disulfonate (BCS) [6] has been successfully utilized to characterize cuproproteins with attomolar dissociation constants, [2b] as a bidentate ligand it must be employed at significant excess over Cu I and in some cases ternary complex formation has been reported. [6–7] Synthetic ligands with sub-attomolar dissociation constants have been realized by mimicking the thiolate coordination environment of Cu I proteins; [8] however, thiolates may also bind to other biologically relevant metal cations such as Zn II or Fe II and are sensitive to pH and thiol-disulfide redox status, both of which may vary with the cellular microenvironment.…”

“…Membrane-permeant ligands that selectively bind to am etal ion of interest represent ac ritical tool for interrogating metal homeostasis; [4] however, the affinity of most currently available Cu I ligands does not match the biologically important attomolar buffer window.For example,mixed amine-thioether donors,w hich have been frequently employed for the construction of Cu I -selective fluorescent probes, [5] offer dissociation constants in the pico-to femtomolar range.W hile bathocuproine disulfonate (BCS) [6] has been successfully utilized to characterize cuproproteins with attomolar dissociation constants, [2b] as ab identate ligand it must be employed at significant excess over Cu I and in some cases ternary complex formation has been reported. [6,7] Synthetic ligands with sub-attomolar dissociation constants have been realized by mimicking the thiolate coordination environment of Cu I proteins; [8] however, thiolates may also bind to other biologically relevant metal cations such as Zn II or Fe II and are sensitive to pH and thiol-disulfide redox status, both of which may vary with the cellular microenvironment.…”

Exploring the role of the various methionine residues in the Escherichia coli CusB adapter protein

“…Cu(I)-(BCA) 2 shows a characteristic high absorption at 562 nm. The binding affinity of proteins for Cu(I) can be calculated by monitoring the change in the absorption peak intensity of the Cu(I)-(BCA) 2 complex at 562 nm, which is associated with protein titration [42, 43].…”

“…To derive the K D values, we used β 2 = 10 17.2 M −2 [42, 43]. The calculated K D values are listed in Table 1:…”

Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

“…However, more recent data using a series of peptides coupled to dansyl fluorophores validated our results showing nanomolar binding affinities of copper to the E1 domain, but now indicated a picomolar affinity of the M1 site in the E2 domain of APP (Young et al . ). Those studies were conducted with the individual domains (E1, E2, GFLD and CuBD) as well as sAPPα in the presence of heparin.…”