470 | Metallomics, 2020, 12, 470--473 This journal is © The Royal Society of Chemistry 2020 Cite this: Metallomics, 2020, 12, 470 Hierarchical binding of copper II to N-truncated Ab 4–16 peptide† Xiangyu Teng, ‡ a Ewelina Stefaniak, ‡ b Paul Girvan, a Radosław Kotuniak, b Dawid Płonka, b Wojciech Bal * b and Liming Ying * c N-Truncated Ab 4–42 displays a high binding affinity with Cu II .A mechanistic scheme of the interactions between Ab 4–42 and Cu II has been proposed using a fluorescence approach. The timescales of different conversion steps were determined. This kinetic mechanism indicates the potential synaptic functions of Ab 4–42 during neuro- transmission. The amyloid-b (Ab) peptides associated with Alzheimer’s Disease (AD) comprise a number of species. The ‘‘canonical’’ Ab 1–42 and Ab 1–40 peptides derived directly by proteolysis of the Amyloid Precursor Protein (APP) are complemented by N- and C-truncated species, yielded by a variety of brain proteases. 1 Among them, the N-truncated Ab 4–42 has been reported as particularly abundant in the hippocampus and cortex of sporadic AD patients, as well as in healthy controls, 2,3 even exceeding Ab 1–42 and Ab 1–40 . 4,5 Ab 1–x peptides can bind Cu II using the N-terminus and H6, H13, and His14 residues. 6–8 Hence, Ab 1–16 has been adopted as a common model peptide in metal binding studies. K d in the range of 0.1 nM to 1 nM at pH 7.1–7.4 was determined for Ab 1–16 and Ab 1–40 . 9–11 The adventitious binding of Cu II ions to Ab 1–42/40 and the concomitant generation of reactive oxygen species (ROS) via the Cu II /Cu I redox pair has been proposed to be the molecular basis of oxidative stress and neuronal death in AD. 12 On the other hand, Ab 4–x peptides bind a Cu II ion more than three orders of magni- tude more strongly (K d = 30 fM and 6.6 fM at pH 7.4 for Ab 4–16 and Ab 4–9 , respectively), using their N-terminal ATCUN motif spanning the Phe4, Arg5 and His6 residues. These complexes are redox-inert and do not generate significant ROS. Cu II ion transfer from Ab 1–16 to Ab 4–16 occurs upon adding the latter to the Cu II Ab 1–16 solution. 13 This reaction is quantitative, in agreement with the affinity difference, and fast, occurring within the sample preparation time Bs. Such a reaction suggested that Ab 4–42 should prevail as a Cu II binding Ab species in the extracellular spaces of the brain. This finding gave rise to a hypothesis that Ab 4–42 may have a physiolo- gical role as a synaptic Cu II scavenger during neurotransmission. 14 However, Cu II release events in glutamatergic synapses may occur on a much faster, millisecond scale. Therefore, a thorough deter- mination of association and dissociation rate constants for the participating species is necessary to help evaluate their relevance in vivo. Such data have been obtained previously for Cu II Ab 1Àx complexes. 15–17 Here, we studied the reaction mechanism for Cu II binding to the model peptide Ab 4–16 and found that the reaction follows a hierarchical fashion, going through two intermediate states and then reaching the final tight complex. First, we studied the effect of N-truncation on the Cu II binding kinetics. 20 nM Ab labelled by HiLyte Fluor 488 on lysine 16 (FRHDSGYEVHHQK-HiLyte 488) was reacted with 400 nM Cu II under various HEPES concentrations in order to obtain the HEPES-independent Cu II binding rate constant (k on ). The results are shown in Fig. 1a. The intercept of the fitted curve (Fig. 1b) was used to determine k on , which is 2.0(1) Â 10 8 M À1 s À1 , 2.5 times slower than the value for Ab 1–16 . 17 k off was determined for the reaction of a Cu II complex of unlabelled Ab 4–16 with an excess of EDTA. The estimated value a Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, White City Campus, London W12 0BZ, UK b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin ´skiego 5a, 02-106 Warsaw, Poland. E-mail: wbal@ibb.waw.pl c National Heart and Lung Institute, Imperial College London, Molecular Sciences Research Hub, White City Campus, London W12 0BZ, UK. E-mail: l.ying@imperial.ac.uk † Electronic supplementary information (ESI) available. See DOI: 10.1039/c9mt00299e ‡ These authors contributed equally to this work. Received 7th December 2019, Accepted 18th February 2020 DOI: 10.1039/c9mt00299e rsc.li/metallomics Significance to metallomics N-Truncated Ab 4–x is abundant in both healthy and AD brains. Its Cu(II) binding affinity is three orders of magnitude stronger than well-known Ab 1–42 or Ab 1–40 . Using a model peptide, Ab 4–16 , we have elucidated the reaction mechanism of Cu(II) with Ab 4–x , crucial to understand the physiological role and toxicity of Ab peptides. The presence of two kinetic intermediates prior to the formation of the tight ATCUN complex has implications for the potential function of Ab 4–42 as a Cu(II) transporter during neurotransmission. The methodology used in this work may also stimulate the research of Cu(II) interactions with other intrinsically disordered proteins (IDPs). Metallomics COMMUNICATION Downloaded from https://academic.oup.com/metallomics/article/12/4/470/5961792 by guest on 02 March 2023