Hierarchical binding of copper II to N-truncated A b 4–16 peptide †

N-Truncated A b 4–42 displays a high binding affinity with Cu II . A mechanistic scheme of the interactions between A b 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 A b 4–42 during neurotransmission. N-Truncated A b 4– x is abundant in both healthy and AD brains. Its Cu( II ) binding affinity is three orders of magnitude stronger than well-known A b 1–42 or A b 1–40 . Using a model peptide, A b 4–16 , we have elucidated the reaction mechanism of Cu( II ) with A b 4– x , crucial to understand the physiological role and toxicity of A b peptides. The presence of two kinetic intermediates prior to the formation of the tight ATCUN complex has implications for the potential function of A b 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).

binding Ab species in the extracellular spaces of the brain. This finding gave rise to a hypothesis that Ab 4-42 may have a physiological 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 determination 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][16][17] Here, we studied the reaction mechanism for Cu II binding to the model peptide Ab [4][5][6][7][8][9][10][11][12][13][14][15][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 is B5 Â 10 À5 s À1 , which divided by k on proposed here gives K d B 250 fM. EDTA is a stronger Cu II chelator than Ab 4-16 , with a log b of 18.7, which can be recalculated into a conditional constant C K of 16.0 at pH 7.5. 18 This value is sufficiently higher than that of Cu II Ab 4-16 , 13.53, to assure full Cu II transfer, as demonstrated in Fig. 1c. The reaction was carried out for a range of EDTA/peptide ratios between 2 and 120. Pseudo-1st order kinetics for the Cu II transfer reaction was observed for all experiments. The non-linear response of k off to EDTA required the EDTA-independent k off value to be determined by the extrapolation of the empirical exponential fit to these data, as shown in Fig. 1d.
To gain a glimpse of a possible reaction mechanism of Cu II binding to N-truncated Ab 4-16 , we performed binding experiments at a 1 : 1 mixing ratio of Ab to Cu II with increasing concentration. In such experiments, the effect of the second Cu II binding can be ignored, as the relevant log K is as low as 6.7. 13 The raw traces are shown in Fig. 2a. We noticed that the reaction process is becoming concentration independent after B2 s (results from the fit are summarized in Table S1, ESI †). Thus we infer the existence of an intramolecular process following the initial Cu II binding.
Next, a double mixing stopped flow technique was employed to further explore the potential intermediate complexes formed after the initial Cu II binding. This technique was successfully applied to probe the interconversion between component I and component II Cu II coordination species of Ab 1-16 and Ab 1-40 . 17 2 mM Ab 4-16 and 2 mM Cu II were mixed in a delay loop and after various delay times the reaction was ''frozen'' by adding an excess of EDTA (Fig. 2b). Taking advantage of the disparities in reactivity of different Cu II Ab 4-16 species with EDTA, the time evolution of the population of individual species could be resolved and analyzed, enabling us to depict details of the binding process.
As shown in Fig. 2b, the amplitude of fluorescence recovery strongly depends on the delay time, indicating that a much more inert (less reactive towards EDTA) complex (''dark'' complex) formed after around 2 s. We equate this end complex, (Ab-Cu) D , to the very stable ATCUN-type Cu II Ab 4-16 complex reported previously. 13 Furthermore, because the reaction rate is concentration independent after 2 s as mentioned above, we propose that a peptide conformational rearrangement process leading to this final complex must occur at around 2 s.
In order to describe the whole process of Cu II binding of N-truncated Ab 4-16 , we hypothesized a reaction scheme as shown in Fig. 3a. The individual amplitudes of the two phases in Fig. 2b were determined by a global fit, which were further fitted by the scheme with KinTek software to validate it (Fig. 3b). The amplitudes indicate the amounts of two intermediates, Species I and Species II, at different reaction process stages, and could be   fitted well by the predicted mechanism, with fitted rate constants listed in Table 1. A corresponding free energy landscape illustration of Cu II binding with Ab 4-16 is shown in Fig. 3c.
Finally, the activation energy of the (Ab-Cu) D complex was determined to be 64(3) kJ mol À1 (Fig. 4) by performing a series of double mixing experiments at different temperatures (raw data shown in Fig. S1, ESI †).
The chemical properties of ATCUN Cu II complexes of Ab 4-x peptides, such as high thermodynamic stability, absence of ROS production due to their resistance to oxidation and reduction, reluctance of copper to transfer to metallothionein-3 (MT3) and easy sequestering of Cu II from Ab 1-x , gave rise to a concept that Ab 4-x peptides (full-length Ab 4-42 and its C-truncated analogs) may serve as guardians of synaptic function, by sequestering excess Cu II ions released during neurotransmission in glutamatergic pathways. 14,19 The key unsolved issue is how these exchange-inert complexes relay copper back to neurons to maintain the proper copper cycling. Furthermore, Cu II -free Ab 4-42 can be neurotoxic by forming oligomeric species. 20 Detailed knowledge on mechanisms of Cu II association with and dissociation from Ab 4-x peptides, represented here by Ab 4-16 , is thus crucial to understand the physiology and toxicity of these Ab peptides.
The discovery of long-lived kinetic intermediates in the formation of the ATCUN complex of Ab 4-16 is a game changer in the above considerations. The lifetimes of Species I and Species II complexes are comparable to the intervals between pulses of neurotransmitter release in glutamatergic neuronal pathways. 21 Therefore, these complexes may well contribute to the biological activity of Ab 4-42 , and of putative short peptidic fragments generated by neprilysin cleavage, such as Ab 4-9 . 22,23 There is only one way in which four nitrogen ligands of the ATCUN motif can be arranged around the Cu II ion, and so it is reasonable to assume that the intermediate species contain the coordinatively unsaturated Cu II . Such species have been implicated in the reverse reaction of Cu II dissociative transfer from Cu II Ab 4-16 to MT3, to explain the catalytic effect of glutamate, 24 but it has not been observed directly. The Species I and in particular the longer-lived Species II complex may be the actual species able to move copper around during neurotransmission. The fact that the Cu II Ab 1Àx complex, although so much weaker, was formed 2.5 times faster, prompts further research into possible synaptic roles of Cu II interactions with various Ab species.
Furthermore, the observed hierarchical binding of Cu II to Ab 4-16 resembles the kinetics of the binding of many intrinsically disordered proteins (IDPs). 25 The methodology used in this study may be applicable to the fundamental understanding of the emerging ''coupled binding and folding'' paradigm. 26

Conflicts of interest
There are no conflicts to declare. Table 1 Rate constants corresponding to the mechanism scheme shown in Fig. 3a k +1 k À1 k +2 k value/s À1 4.10(1) 10.34 (2) 3.31(4) Fig. 4 Arrhenius plot for the switching rate constant k +2 . The switching activation energy determined is 64(3) kJ mol À1 .