Hexa-Histidine, a Peptide with Versatile Applications in the Study of Amyloid-β(1–42) Molecular Mechanisms of Action

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1–42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer’s disease patients. Aβ(25–35) is the shortest peptide that retains the toxicity of Aβ(1–42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1–42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1–42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1–42). The formation of His6/Aβ(1–42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1–42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1–42) and Aβ(25–35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

Aβ peptides bind to ganglioside-clustered raft-like membrane microdomains, which foster the formation of Aβ oligomers and fibrils in a cholesterol-dependent manner [18][19][20][21][22]. Indeed, dimeric nonfibrillar Aβ has been shown to accumulate in lipid rafts in the Tg2576 mouse model of AD [23], and exogenous oligomeric Aβ applied to neurons in culture concentrates in lipid rafts [24].Moreover, plasma membrane lipid rafts play an active role in extracellular Aβ uptake and internalization in neurons [25].After the uptake of extracellular Aβ(1-42) into neurons, the intracellular aggregates elicit neuronal damage and neurotoxicity [8,9,26,27].Intracellular targets of Aβ(1-42) oligomers have been identified in the cytosol and subcellular organelles like the endoplasmic reticulum (ER) and mitochondria [28][29][30].Of particular relevance are the intracellular targets of Aβ(1-42) oligomers that bind nanomolar concentrations of Aβ peptides, because concentrations of non-fibrillar Aβ peptides within the nanomolar range have been reported in the brain [31][32][33], and the critical concentration that induces Aβ(1-42) fibrillization lies in the submicromolar range [34,35].Until now, dissociation constants lower than 10 nM have been reported for the complexes between Aβ peptides and a few intracellular proteins expressed in brain neurons, namely calmodulin (CaM) [36], cellular prion protein [37], glycogen synthase kinase 3α [38], tau [39], calbindin-D28k [40] and STIM1 [30].However, in neurons, the concentration of CaM is in the micromolar range, which is orders of magnitude higher than the concentration of the other competing proteins in neurons listed above, with the exception of calbindin-D28k.Nevertheless, it is to be noted that, although calbindin-D28k is abundant throughout the central nervous system, including pyramidal hippocampal neurons and cortical neurons [41], the dissociation constant of the Aβ(1-42)/calbindin-D28k complex is around 10-fold higher than that of the Aβ(1-42)/CaM complex [40].Due to this, CaM binds a large fraction of intracellular Aβ  in the nanomolar concentration range [27].
In this work, we report that the fluorescence of Aβ(1-42) HiLyte TM -Fluor555 (dye covalently bound to ASP1 of Aβ) monitors hexa-histidine peptide (His 6 ) interaction with Aβ(1-42) and the inhibition of the interaction between Aβ(1-42) and the proteins CaM and calbindin-D28k.Also, we found that the presence of a C-terminal His 6 -tag in human recombinant cytochrome b 5 reductase (Cb5R) sensitizes its enzymatic activity to modulation by submicromolar Aβ(1-42) concentrations.In this work, we have used human recombinant Cb5R because the cytochrome b 5 (Cb 5 )/Cb5R system has a relevant role in lipid metabolism, which is significantly altered in AD [56,57].

Hexa-Histidine (His 6 ) Interacts with Aβ(1-42) HiLyte TM -Fluor555
The addition of submicromolar concentrations of His 6 to a solution of 10 nM Aβ(1-42) HiLyte TM -Fluor555 increases the fluorescence intensity of the labelled peptide (Figure 1A).The kinetics of the increase in fluorescence, which monitors the interaction between His 6 and Aβ(1-42) HiLyte TM -Fluor555, has an initial slower phase of 1-2 min and reaches a maximum of 8 ± 2% with a half-time of approximately 10 min.Although the kinetics are slightly faster with 0.25 µM of His 6 , the large overlap between the kinetic traces obtained with 0.1 and 0.25 µM of His 6 points out that at the latter concentration, no new complexes are being formed.The results of titration with His 6 shown in Figure 1B confirm the saturation of the maximum fluorescence change at a concentration of 0.1 µM of His 6 .The small value of the maximum fluorescence change indicates that His 6 interaction with Aβ(1-42) elicits only a small change in the microenvironment of the HiLyte TM -Fluor555 dye, which is bound to ASP1 of Aβ .The short initial lag phase suggests that conformational changes in His 6 and/or Aβ(1-42) HiLyte TM -Fluor555 are needed for a better coupling between both molecules.The dependence upon the His 6 concentration of the increase in fluorescence intensity at 2000 s of the kinetic of the increase in fluorescence is shown in Figure 1B.Taking into account that the noise of the fluorescence signal introduces an error close to ±2% in the differences between fluorescence intensity readings, the 50% of the maximum fluorescence change is reached with a concentration of ≈50-60 nM of His 6 , i.e., the saturation of the fluorescence change is observed at ratios ≥10 molecules of His 6 per Aβ(1-42) HiLyte TM -Fluor555 monomer.

Hexa-Histidine (His6) Interacts with Aβ(1-42) HiLyte TM -Fluor555
The addition of submicromolar concentrations of His6 to a solution of 10 nM Aβ(1-42) HiLyte TM -Fluor555 increases the fluorescence intensity of the labelled peptide (Figure 1A).The kinetics of the increase in fluorescence, which monitors the interaction between His6 and Aβ(1-42) HiLyte TM -Fluor555, has an initial slower phase of 1-2 min and reaches a maximum of 8 ± 2% with a half-time of approximately 10 min.Although the kinetics are slightly faster with 0.25 µM of His6, the large overlap between the kinetic traces obtained with 0.1 and 0.25 µM of His6 points out that at the latter concentration, no new complexes are being formed.The results of titration with His6 shown in Figure 1B    The experimental results shown above prompted us to perform docking between His 6 and/or Aβ .The analysis of docking has been performed using the MDockPeP Server, as indicated in Section 4. The structure of Aβ(1-42) registered with PDB ID: 1IYT in UniPro Databank has been selected for this docking work due to the following reasons: (1) at the concentration of 10 nM used in these experiments, the Aβ(1-42) peptide must be largely in the monomeric state because the dissociation constant for Aβ(1-42) dimers is similar to that reported for Aβ(1-40) dimers, i.e., around 198 ± 43 nM [58], and (2) we found in a previous work that this structure of the Aβ(1-42) monomer is a good template structure for the design of an efficient peptide antagonist for the interaction between Aβ(1-42) and CaM [40].The results generated 10 possible structural models for the complex formation, which can be grouped into three clusters, as shown in Table 1 and Supplementary Tables.In cluster 1, His 6 interacts with the Aβ(1-42) domain close to the NH 2 -terminus and involves the short α-helix and the part of the long α-helix near the structural tilt of this peptide.Cluster 1 gathers models 1, 4, 7 and 8 and presents an estimated free energy for the complex formation of −6.8 kcal/mol, which is that found for the lowest energy model of the cluster (model 7) (Figure 2, cluster 1).The interacting interface in cluster 1 comprises the following amino acid residues of the Aβ peptide: LYS16, LEU1, PHE20, ALA21, VAL24, GLY25, LYS2, ILE31, LEU34 and MET35 and HIS1, HIS3, HIS4, HIS5 and HIS6 residues of the His 6 peptide.Buried surface area/accessible solvent area ratio allowed us to rank those amino acid residues with higher participation in the interaction with Aβ peptide: VAL24 > ALA21 > GLY25 > LEU34 > PHE20 > MET35 > LEU17 > ILE31 > LYS28 > LYS16 and a similar participation for the HIS1, HIS3, HIS4, HIS5 and HIS6 of the His 6 peptide.Several H-bonds and salt bridges were found in models from cluster 1 but without sharing the same amino acid residues among themselves (Supplementary Table S1).
Cluster 2 is the second most probable cluster and is representative of another four of the ten most probable outcomes generated using the MDockPeP Server.In cluster 2, His 6 interacts with the Aβ(1-42) domain close to the COOH-terminus.Cluster 2 gathers models 2, 5, 9 and 10.Analysis with PDBePisa estimated free energy for the complex formation of −4.8 kcal/mol, which is that found for the lowest energy model of the cluster (model 9) (Figure 2, cluster 2).The interacting interface in cluster 2 comprises the following amino acid residues of the Aβ peptide: GLU3, HIS6, ASP7, TYR10, HIS14 and HIS1, HIS2, HIS6 residues of the His 6 peptide.Buried surface area/accessible solvent area ratio allowed us to rank those amino acid residues with higher participation in the interaction with Aβ peptide: TYR10 > HIS6 > GLU3 > HIS14 > ASP7 and similar participation for the HIS1, HIS2 and HIS6 of the His 6 peptide.Several H-bonds and salt bridges were also found in models from cluster 2, but as also accounting for cluster 1, they did not share similarities among themselves (Supplementary Table S2).A third cluster was found based on models sharing similarities in their interaction.Cluster 3 is representative of two of the ten most probable structures generated using the MDockPeP Server and predicts that His 6 may also interact with the long α-helix part closer to the COOH-terminus of Aβ .Cluster 3 gathers models 3 and 6. Analysis with PDBePisa estimated free energy for the complex formation of −2.6 kcal/mol, which is that found for the lowest energy model of the cluster (model 3) (Figure 2, cluster 3).The interacting interface in cluster 3 comprises the GLY9 Molecules 2023, 28, 7138 5 of 20 and VAL184 amino acid residues of Aβ peptide and HIS1, HIS2, HIS6 residues of the His 6 peptide.Buried surface area/ accessible solvent area ratio indicates that both amino acid residues of Aβ peptide (GLY9 and VAL184) participate similarly in the interaction with the His 6 peptide and Aβ peptide.Several H-bonds and salt bridges were also found in models from cluster 3 without sharing similarities among themselves (Supplementary Table S3).Cluster 2 is the second most probable cluster and is representative of another four of the ten most probable outcomes generated using the MDockPeP Server.In cluster 2, His6 interacts with the Aβ(1-42) domain close to the COOH-terminus.Cluster 2 gathers models 2, 5, 9 and 10.Analysis with PDBePisa estimated free energy for the complex formation of −4.8 kcal/mol, which is that found for the lowest energy model of the cluster (model 9) (Figure 2, cluster 2).The interacting interface in cluster 2 comprises the following amino acid residues of the Aβ peptide: GLU3, HIS6, ASP7, TYR10, HIS14 and HIS1, HIS2, HIS6 residues of the His6 peptide.Buried surface area/accessible solvent area ratio allowed us to rank those amino acid residues with higher participation in the interaction with Aβ peptide: TYR10 > HIS6 > GLU3 > HIS14 > ASP7 and similar participation for the HIS1, We can summarize that the analysis in silico allows us to predict large conformational plasticity in the His 6 /Aβ(1-42) complex, suggesting that the predominant conformation of this complex could be strongly affected by its microenvironment.In addition, docking results raise the possibility that the Aβ(1-42) monomer can bind up to 2-3 molecules of His 6 .

His 6 Antagonizes the Interaction of Aβ(1-42) HiLyte TM -Fluor555 with CaM and Calbindin D28k
In previous works, we have shown that Aβ(1-42) binds with high affinity to CaM and calbindin-D28k [36,40].Since model 1 predicts a strong interaction of His 6 with the domain of Aβ(1-42) more directly involved in its interaction with CaM and calbindin-D28k, in this work, we have experimentally assessed the possibility that His 6 could antagonize the interaction of Aβ(1-42) with these proteins.
The kinetics of the interaction of the fluorescence derivative of Aβ(1-42), Aβ(1-42) HiLyte TM -Fluor555, with CaM and calbindin-D28k can be monitored by the fluorescence intensity changes of the dye HiLyte TM -Fluor555 [40].A preincubation of 15 min with His 6 was included in the experimental design to prevent a distortion of the kinetics of fluorescence increase produced by CaM or calbindin-D28k by the change of fluorescence elicited by His 6 .Figure 3 shows that His 6 attenuates the increase in fluorescence intensity Aβ(1-42) HiLyte TM -Fluor555 after adding either calbindin-D28k or CaM.This is an effect dependent upon the concentration of His 6 .As shown in Figure 3, the maximum change of the fluorescence intensity and the rate of the kinetic process are decreased by His 6 , albeit with different efficacy.These results yield His 6 concentrations for a 50% attenuation of the maximum change of fluorescence intensity of ≈0.1 and 1 µM for the interaction of Aβ(1-42) with calbindin-D28k and CaM, respectively.Therefore, His 6 behaves as an antagonist of the interaction of Aβ(1-42) with these proteins, although with different potency in the submicromolar-to-micromolar concentration range.In order to evaluate the ability of His 6 to sensitize proteins to Aβ peptides, we have used human recombinant Cb5R with a His 6 -tag covalently linked to the NH 2 -terminus amino acid, expressed and purified as described in Section 4. The NADH-dependent reduction in Cb 5 has been measured, following protocols used in previous works, as indicated in Section 4.

Submicromolar Concentrations of Aβ Peptides
Representative kinetic traces of the rate of NADH-dependent reduction in Cb 5 are presented in Figure 4A.A specific activity of 7.9 ± 0.7 µmoles of the reduced Cb 5 /min/mg of Cb5R at 25 • C has been calculated from initial rate measurements for the samples of human recombinant C-terminal His 6 -tagged Cb5R.Also, the kinetic traces shown in Figure 4A highlight the potent inhibition of this activity by nanomolar Aβ(1-42) concentrations.As up to 2 µM of Aβ(1-42), the highest concentration tested in this work, we have not detected any alteration of the absorbance spectrum of Cb 5 , the possibility that this inhibition could be due to an Aβ(1-42)-induced conformational change in the microenvironment of the heme group of Cb 5 can be discarded.Moreover, the inhibition is seen from the first absorbance measurements after the addition of Aβ(1-42), implying its rapid binding to the inhibitory site in the Cb5R.Indeed, the observed inhibition was the same without or with up to 30 min preincubation of the Cb5R with 50 and 100 nM of Aβ(1-42).Next, we removed the His 6 -tag from human recombinant Cb5R as described in detail in Section 4. We found that the addition of up to 2 µM of Aβ(1-42) and of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) does not produce a statistically significant inhibition of the rate of NADH-dependent reduction in Cb 5 by human recombinant Cb5R minus a His 6 -tag, i.e., less than 10% inhibition.Of note, a 2 micromolar concentration of Aβ(1-42) oligomers is higher than those that can be expected intracellularly in AD, since the induction of fibrillization only requires submicromolar concentrations of Aβ(1-42) [34,35].

Submicromolar Concentrations of Aβ Peptides Inhibit the Reduction in Cb5 Catalyzed by Purified Recombinant His6-Tagged Cb5R, and No Inhibition Is Observed after Deletion of the His6-Tag
In order to evaluate the ability of His6 to sensitize proteins to Aβ peptides, we have used human recombinant Cb5R with a His6-tag covalently linked to the NH2-terminus amino acid, expressed and purified as described in the Materials and Methods section.The NADH-dependent reduction in Cb5 has been measured, following protocols used in previous works, as indicated in the Materials and Methods section.
Combining these kinetic results, it merges the conclusion that the Cb5 reductase activity of the His6-tagged Cb5R is strongly sensitized to neurotoxic Aβ peptides.Combining these kinetic results, it merges the conclusion that the Cb 5 reductase activity of the His 6 -tagged Cb5R is strongly sensitized to neurotoxic Aβ peptides.(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) to the Human Recombinant Cb5R with a His 6 -tag Increases the Fluorescence of Its Prosthetic Group FAD In previous works [59,60], we have shown that the fluorescence of the prosthetic group FAD can be used to monitor conformational changes of Cb5R, which impair its catalytic activity.

The Binding of Aβ(1-42) and of Aβ
In this work, we found that the addition of Aβ(1-42) and of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) produces an increase in the FAD fluorescence of human recombinant Cb5R with a His 6 -tag without a significant shift of the emission peak wavelength due to a conformational change that leads to an increase in the quantum yield of FAD bound to the Cb5R. Figure 5 shows that the FAD fluorescence increase depends on the concentration of the Aβ(1-42) and of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) peptides.Moreover, both the magnitude and the dependence upon the Aβ peptide concentration of the change of FAD fluorescence are not significantly different between Aβ(1-42) and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).Of note, this is an effect produced by the assayed neurotoxic peptides because up to 1 µM of "scrambled" Aβ(1-42) peptide does not elicit a statistically significant change in FAD fluorescence.The results obtained for the dependence of the increase in FAD fluorescence upon the concentration of Aβ(1-42) and of Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fit well into the equation for one-site ligand-protein interaction (Figure 5).The non-linear fit of the results to this equation yields an increase of 41 ± 4% of the FAD fluorescence at saturation with these peptides, and an Aβ concentration for 50% of the effect of 75 ± 5 nM of peptide monomer.Since the concentration of Cb5R in the fluorescence assays is 32 ± 3 nM, the real free-peptide concentration at 50% saturation allows us to calculate an apparent dissociation constant (K d,app ) of 59 ± 5 nM for the complex between the Cb5R and the Aβ peptide.This K d,app value is within the range of values obtained for the Ki, pointing out that the binding of the Aβ peptide to this site causes the inhibition of the NADH-dependent Cb 5 reductase activity of the human recombinant Cb5R with a His 6 -tag.Since the dependence of the FAD fluorescence upon the concentration of Aβ(1-42) was the same in the absence and presence of 5 µM of Cb 5 , the possibility that Aβ could interact with the Cb 5 binding domain in the Cb5R can be excluded.Also, this result indicates that Aβ(1-42) does not bind to Cb 5 , resulting in further experimental support to our conclusion derived from the lack of effect of Aβ(1-42) on the absorbance spectrum of Cb 5 (see above).
ent Cb5 reductase activity of the human recombinant Cb5R with a His6-tag.Since the dependence of the FAD fluorescence upon the concentration of Aβ(1-42) was the same in the absence and presence of 5 µM of Cb5, the possibility that Aβ could interact with the Cb5 binding domain in the Cb5R can be excluded.Also, this result indicates that Aβ(1-42) does not bind to Cb5, resulting in further experimental support to our conclusion derived from the lack of effect of Aβ(1-42) on the absorbance spectrum of Cb5 (see above).In order to further assess the interaction between Aβ(1-42) and human recombinant Cb5R with a His 6 -tag, we have used the fluorescent derivative Aβ(1-42) HiLyte TM -Fluor555.This compound can act as a fluorescence resonance energy transfer (FRET) acceptor of FAD fluorescence as illustrated by the overlap between the emission fluorescence spectrum of FAD and the absorbance spectrum (Figure 6A).Next, we calculated the distance for 50% FRET efficiency (R 0 ) between FAD and Aβ(1-42) HiLyte TM -Fluor555 as described in Section 4, following a protocol used in previous works [61][62][63].A value of 3.96 × 10 −3 has been calculated for the quantum yield of FAD bound to the Cb5R (Φ D ), using as reference the quantum yield of FMN given in standard fluorescence data tables.A value of the overlap integral J(λ) of 6.0254174 × 10 15 cm 3 •M −1 has been calculated from the recorded emission spectrum of FAD bound to the Cb5R and the absorbance spectrum of Aβ(1-42) HiLyte TM -Fluor555.An R 0 value of 2.77 nm has been obtained by applying the equation given in Section 4, assuming a random orientation between donor and acceptor.
Then, we recorded the emission spectrum of human recombinant Cb5R with a His 6 -tag in the absence and presence of 100 nM of Aβ(1-42) HiLyte TM -Fluor555 and the emission spectrum of 100 nM Aβ(1-42) HiLyte TM -Fluor555 in the absence of Cb5R (Figure 6B).Next, the latter spectrum, which is due to the direct excitation of the acceptor HiLyte-Fluor555, has been subtracted to the emission spectrum of Cb5R plus 100 nM of Aβ(1-42) HiLyte TM -Fluor555, and the result is shown in Figure 6C.We avoided the use of a saturating concentration of the Aβ(1-42)-induced increase in the FAD fluorescence in Cb5R shown in Figure 5 to prevent the complications of analysis coming from an excessively large peak of the HiLyte TM -Fluor555 fluorescence on the overall emission spectrum.The results shown in Figure 6C indicate that 100 nM of Aβ(1-42) HiLyte TM -Fluor555 produces a 45 ± 5% quenching of the peak donor FAD fluorescence at 525 nm, and a similar increase in the peak of the acceptor HiLyte TM -Fluor555 fluorescence at 555 nm.Thus, FRET measurements provide an additional proof of interaction between Cb5R with a His 6 -tag and Aβ(1-42) HiLyte TM -Fluor555.
shown in Figure 5 to prevent the complications of analysis coming from an excessively large peak of the HiLyte TM -Fluor555 fluorescence on the overall emission spectrum.The results shown in Figure 6C indicate that 100 nM of Aβ(1-42) HiLyte TM -Fluor555 produces a 45 ± 5% quenching of the peak donor FAD fluorescence at 525 nm, and a similar increase in the peak of the acceptor HiLyte TM -Fluor555 fluorescence at 555 nm.Thus, FRET measurements provide an additional proof of interaction between Cb5R with a His6-tag and Aβ(1-42) HiLyte TM -Fluor555.

Discussion
The results show the interaction between Aβ(1-42) HiLyte TM -Fluor555 and submicromolar concentrations of His 6 .Likely, this interaction will be further potentiated by transition metal ions, which bind with very high affinity to Aβ(1-42) [2,54], and, also, to His 6 [64].Indeed, we cannot completely exclude that trace amounts of transition metal ions, which are present in the preparations of commercial samples of Aβ peptides and in buffers, are playing a role in the Aβ(1-42) HiLyte TM -Fluor555/His6 complexes formed under our experimental conditions.The kinetics of the increase in Aβ(1-42) HiLyte TM -Fluor555 fluorescence intensity after the addition of His 6 is a relatively slow process for this type of molecules, with a half-time of around 10 min, and an initial short lag phase within the first minute.Therefore, the kinetic results suggest the occurrence of conformational changes in the formation of a complex between Aβ(1-42) HiLyte TM -Fluor555 and His 6 .Also, it is to be noted that a concentration of 100 nM His 6 is almost saturating for its complexation with 10 nM Aβ(1-42) HiLyte TM -Fluor555, indicating that the dissociation constant (Kd) of this complex is in the submicromolar concentration range.However, the maximum increase in the Aβ(1-42) HiLyte TM -Fluor555 fluorescence intensity (<10%) is not large enough over the noise of the data to obtain a reliable Kd value for this complex from the dependence upon the His 6 concentration using this experimental approach.Docking simulations provide a rational support for the formation of a complex between His 6 and Aβ .The simulations performed using the CABS-dock Web Server, which do not require the input of an initial conformation of His 6 , yield three-dimensional structures for this complex that can be grouped in three-classes taking into account the Aβ(1-42) peptide domain directly interacting with His 6 .Thus, changes in the structure of His 6 are one likely cause of the short initial lag phase of the kinetics of complex formation.On the other hand, the cluster of poses represented by cluster 1 of Figure 2, generated using docking simulations for the His 6 /Aβ(1-42) complex, predicts that His 6 interacts with amino acid residues 24-42 of Aβ .In a previous work [40], we have shown that this domain of Aβ(1-42) is the most directly involved in the formation of Aβ(1-42)/CaM and Aβ(1-42)/calbindin-D28k complexes.However, docking analysis predicts a relevant contribution of LYS28 and LYS16 in the case of the cluster 1 of the His 6 /Aβ(1-42) complex, the first cluster ranked by energy calculations, while only highly hydrophobic amino acids of Aβ(1-42) are involved in the interaction interface with CaM and calbindin-D28k [40].The contribution of electrostatic interactions and H-bonds in the interface of interaction between His 6 and Aβ(1-42) is even stronger in the structures of the cluster 2 of His 6 /Aβ(1-42) complex, the second ranked by free energy calculations, as the buried surface area/ accessible solvent area ratio results in the following amino acid residues with higher participation in the interaction with the Aβ peptide: TYR10 > HIS6 > GLU3 > HIS14 > ASP7.Also, the presence of charged amino acids ASP1, GLU3, ARG5, ASP7 and GLU11 in the Aβ(1-42) domain of the interacting interface of cluster 3 of His 6 /Aβ(1-42) complex lends further support to the higher relevance of electrostatic interactions in the formation of this complex.Thus, docking analysis points out a higher contribution of electrostatic interactions and H-bonds in the His 6 /Aβ(1-42) complex than in Aβ(1-42)/CaM and Aβ(1-42)/calbindin-D28k complexes.Consistent with this analysis, in this work, we show that His 6 efficiently antagonizes the interaction between Aβ(1-42) HiLyte TM -Fluor555 and these two proteins, with concentrations for 50% effect ≤1 µM of His 6 , values that are more than 10-fold higher than those obtained for the hydrophobic peptide VFAFAMAFML(amidated-C-terminus amino acid) in our previous work [40].However, it is to be noted that the dye HiLyte TM -Fluor555 is covalently bound to the NH 2 -terminus amino acid of Aβ(1-42), which is distant from the amino acid residues 24-42 of Aβ(1-42).Since fluorescence measurements point out that the interaction of His 6 with Aβ(1-42) HiLyte TM -Fluor555 alters the microenvironment of HiLyte TM -Fluor555, it is likely that the structure of the His 6 /Aβ(1-42) complex can be better described by a weighted combination of the three different clusters of His 6 /Aβ(1-42) complex shown in Figure 2 or that the molecular stoichiometry of this complex is higher than 1:1.Indeed, the cluster 2 in Figure 2, the second most probable model yielded by docking simulations, predicts the binding of His 6 to the NH 2 -terminus domain of Aβ(1-42).Nevertheless, the possibility of a significant generation of 3His 6 /Aβ(1-42) complex is unlikely because of the large difference in free energies between clusters 1 and 3, and, also, because of steric hindrance between the His 6 position in clusters 2 and 3.In addition, a shift between cluster structures during the formation of the His 6 /Aβ(1-42) complex could provide a simple explanation for the slow kinetics of interaction between His 6 and Aβ(1-42) HiLyte TM -Fluor555.This is a plausible possibility because docking simulations do not yield large differences of the free energy changes predicted for the clusters 1 and 2 of the His 6 /Aβ(1-42) complex.Further extensive experimental studies will be required to obtain the molecular stoichiometry and structure of the His 6 /Aβ(1-42) complex with atomic resolution.
The large increase in the intensity of the fluorescence of its prosthetic group FAD monitors the binding of Aβ(1-42) and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) to human recombinant Cb5R with a His 6 -tag.Since the catalytic site does not overlap with the position of the His 6 -tag bound to the NH 2 -terminus amino acid of Cb5R, the inhibition by Aβ(1-42) or Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) of its Cb 5 reductase activity is due to a long-distance induced conformational change.Taking into account that 100 nM of Aβ(1-42) HiLyte TM -Fluor555 lies between 60 and 65% of the saturating concentration of Cb5R (Figure 5), we can calculate that at a saturation by Aβ(1-42) HiLyte TM -Fluor555, the quenching of the donor FAD fluorescence will rise up to 70 ± 7%.As at this concentration of Aβ(1-42), the peptide will be in equilibrium between monomers and dimers [58], we have calculated the most probable distance range between FAD and the dye HiLyte TM -Fluor555 using the equation for FRET for 1 and 2 acceptors per donor with the assumption of random orientation given in Section 4. The upper distance limit of this range will be set by the case of the two acceptors HiLyte TM -Fluor555 dyes, one bound to each monomer, located equidistant from the donor FAD.The calculated distances between the donor FAD group of Cb5R and the acceptor HiLyte TM -Fluor555 dye covalently linked to Aβ(1-42) have been 2.35 nm and 3.1 nm for 1 or 2 equidistant acceptors, respectively.Of note, the measurements of the anisotropy of the FAD fluorescence, performed as indicated in Section 4, presented anisotropy values lower than 0.005.As noted in [65], with this low value of donor anisotropy, there is >90% probability that the above-calculated distance range is correct.In order to evaluate whether the structural coupling between the known structures of the Cb5R and Aβ(1-42) allows for the satisfaction of this requirement, we have performed docking between the soluble human erythrocytes isoform of Cb5R (PDB ID: 1UMK) and Aβ(1-42) (PDB ID: 1IYT) using the ClusPro Web Server as described in Section 4.Among the 10 model structures generated in silico, we selected the model structure of the complex between Cb5R and Aβ(1-42) shown in Figure 7, in which the Aβ(1-42) lies close to the His 6 -tag bound to the NH 2 -terminus amino acid of the human recombinant Cb5R.This model structure satisfies the above distance requirement between the NH 2 -terminus amino acid of the Aβ(1-42) and FAD derived from FRET measurements, which can be estimated between 2 and 3 nm by taking into account the uncertainty of the orientation and size of the HiLyte TM -Fluor555 dye in the complex.In addition, this model structure shows that the amino acids 25-35 of the Aβ(1-42) lies near the catalytic center, where the isoalloxazine ring of the FAD prosthetic group of the Cb5R is located.Also, the Cb 5 binding site in the Cb5R, identified in previous works [66,67], lies close to the FAD group in the protein domain located as shown below in Figure 7.The extensive overlap of the 25-35 amino acid residues of the Aβ(1-42) with the Cb 5 domain suggests that steric hindrance and/or an incorrect orientation of bound Cb 5 could account for the inhibition of the Cb 5 reductase activity measured in this work.
model structure shows that the amino acids 25-35 of the Aβ(1-42) lies near the catalytic center, where the isoalloxazine ring of the FAD prosthetic group of the Cb5R is located.Also, the Cb5 binding site in the Cb5R, identified in previous works [66,67], lies close to the FAD group in the protein domain located as shown below in Figure 7.The extensive overlap of the 25-35 amino acid residues of the Aβ(1-42) with the Cb5 domain suggests that steric hindrance and/or an incorrect orientation of bound Cb5 could account for the inhibition of the Cb5 reductase activity measured in this work.In summary, His 6 binds with high affinity to Aβ(1-42), and at micromolar concentrations antagonizes the formation of Aβ(1-42)/CaM and Aβ(1-42)/calbindin-D28k complexes.In addition, a His 6 -tag provides a high-affinity site for the binding of neurotoxic Aβ(1-42) and Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) peptides to the human recombinant Cb5R, and sensitizes this enzyme to inhibition by these peptides.Therefore, our results suggest that a His 6 -tag could be used to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.
All the other chemicals used in this work were of analytical grade and supplied by Merck-Sigma-Aldrich (Madrid, Spain) and ThermoFisher Scientific (Madrid, Spain).

Preparation of the Human Recombinant Cb5R Soluble Isoform
Clones of the soluble isoform of human Cb5R prepared in a previous work were used to express and purify the recombinant protein purified as indicated in [67].The purified protein was aliquoted and conserved in 30% glycerol at −80 • C until use.
The C-terminal His 6 -tag recombinant Cb5R has a thrombin-cutting site between the His 6 -tag and the enzyme, as previously shown [67].The His 6 -tag was cut by overnight incubation at 4 • C with the Thrombin Clean Cleavage TM kit.The recombinant Cb5R minus the His 6 -tag was separated from the cleaved peptide via column chromatography through Sephadex G75 (1 × 25 cm), and the efficient removal of the His 6 -tag was confirmed using SDS-PAGE (Figure 8).

Preparation of the Human Recombinant Calbindin-D28k and Cb5
The human recombinant calbindin-D28k and soluble isoform of Cb5 have been expressed and purified as described in detail in previous works [40,67,69].The purified protein were aliquoted and conserved in 30% glycerol at −80 °C until use.
The concentration of recombinant Cb5R was determined using the method of Bradford with bovine serum albumin as standard or from absorbance measurements at 550 nm using a differential extinction coefficient between reduced and oxidized Cb5 of 16.5 mM −1 •cm −1 , as in previous works [67,69].

Measurements of His6, CaM and Calbindin-D28k Interaction with Aβ(1-42) HiLyte TM -Fluor555
The change of the fluorescence intensity of Aβ(1-42) HiLyte TM -Fluor555 has been used to monitor its complexation with His6, CaM and calbindin-D28k.Fluorescence measurements were performed using a Fluoromax+ fluorescence Spectrophotometer (Horiba Jobin Yvon IBH Ltd., Glasgow, UK) in quartz cuvettes of 1cm light pathlength with a total volume of 2.5 mL at room temperature (24-25 °C), with excitation and emission slits of 5 nm.
The measurements of the kinetics of fluorescence have been performed in buffer 50 mM N- [2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid], 100 mM KCl and 50 µM CaCl2 (pH 7.05).The cuvette was kept with magnetic stirring in the dark within the cuvette holder of the fluorimeter until stabilization of the fluorescence intensity after the addition of 10 nM Aβ(1-42) HiLyte TM -Fluor555, routinely between 20 and 40 min.Then, His6 was added to the cuvette at the concentrations indicated in the figures, and the fluorescence intensity was recorded as a function of time with excitation and emission wavelengths of 520 nm and 567 nm, respectively.His6 was added from stock concentrated solutions prepared in the assay buffer, such that the total added volume was always lower than 10 µL.The concentration of recombinant Cb5R was determined using the method of Bradford, with bovine serum albumin as standard or from absorbance measurements at 450 nm, using an extinction coefficient of 11.3 mM −1 •cm −1 for the FAD prosthetic group [67,68].

Preparation of the Human Recombinant Calbindin-D28k and Cb 5
The human recombinant calbindin-D28k and soluble isoform of Cb 5 have been expressed and purified as described in detail in previous works [40,67,69].The purified protein were aliquoted and conserved in 30% glycerol at −80 • C until use.
The concentration of recombinant Cb5R was determined using the method of Bradford with bovine serum albumin as standard or from absorbance measurements at 550 nm using a differential extinction coefficient between reduced and oxidized Cb 5 of 16.5 mM −1 •cm −1 , as in previous works [67,69]  The change of the fluorescence intensity of Aβ(1-42) HiLyte TM -Fluor555 has been used to monitor its complexation with His 6 , CaM and calbindin-D28k.Fluorescence measurements were performed using a Fluoromax+ fluorescence Spectrophotometer (Horiba Jobin Yvon IBH Ltd., Glasgow, UK) in quartz cuvettes of 1cm light pathlength with a total volume of 2.5 mL at room temperature (24-25 • C), with excitation and emission slits of 5 nm.
The measurements of the kinetics of fluorescence have been performed in buffer 50 mM N-[2-hydroxyethyl] piperazine-N -[2-ethanesulfonic acid], 100 mM KCl and 50 µM CaCl 2 (pH 7.05).The cuvette was kept with magnetic stirring in the dark within the cuvette holder of the fluorimeter until stabilization of the fluorescence intensity after the addition of 10 nM Aβ(1-42) HiLyte TM -Fluor555, routinely between 20 and 40 min.Then, His 6 was added to the cuvette at the concentrations indicated in the figures, and the fluorescence intensity was recorded as a function of time with excitation and emission wavelengths of 520 nm and 567 nm, respectively.His 6 was added from stock concentrated solutions prepared in the assay buffer, such that the total added volume was always lower than 10 µL.Control experiments were run by adding the same volume of the assay buffer, and showed no significant changes in the fluorescence intensity of Aβ(1-42) HiLyte TM -Fluor555.
In the experiments dealing with the effect of His 6 on the kinetics of fluorescence intensity increase after the addition of 5 nM CaM or calbindin-D28k, these proteins were added after 15 min preincubation with the indicated concentration of His 6 .CaM and calbindin-D28k were added from concentrated stock solutions freshly prepared in the assay buffer used in the fluorescence measurements, such that the total added volume was always lower than 10 µL.

In Silico Docking between Aβ(1-42) and His 6
These docking studies have been performed using the MDockPeP Server (https:// zougrouptoolkit.missouri.edu/mdockpep/index.html), accessed on 26 November 2022.This server generates structural simulations of complexes between proteins and peptides, requiring only the input of the PDB file of the protein and the amino acids sequence of the peptide.In this work, we have used the Aβ(1-42) (PDB: 1IYT) as the protein partner.The server generates the 10 more probable poses for the complex formation, on the basis of the results after three major steps: (1) calculation of peptide conformers, in our case, of His 6 ; (2) global flexible sampling of the binding modes protein-peptide; and (3) score and classification of the evaluated types of bonds involved in the formation of the complex [70].Interacting interfaces were analyzed using PDBePISA [71], access date 4-5 October 2023, and they were visualized using UCSF Chimera [72].The measurements were performed with a Shimadzu UV1800 spectrophotometer at a wavelength of 550 nm in 1 mL cuvettes containing the assay buffer 20 mM phosphate/0.1 mM diethylenetriaminepentaacetic acid (pH 7), with 0.11 µg of Cb5R/mL and 5 µM of Cb 5 in the absence and presence of the concentrations of Aβ(1-42), Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and 'scrambled' Aβ(1-42), indicated in the figures.The reaction was started by the addition of 0.25 mM NADH after 10 min preincubation of the Cb5R in the assay medium, and initial rates were measured during periods of time ranging between 5 and 10 min, with a less than 10% drop in the Cb 5 concentration in the assay cuvette.The Cb 5 reductase activity was calculated from the change of absorbance at 550 nm using a differential extinction coefficient between reduced and oxidized Cb 5 of 16.5 mM −1 •cm −1 , as in previous works [67,69].The titration of the NADH-dependent Cb 5 reductase activity of the Cb5R with the Aβ peptide was carried out by measuring in each assay cuvette the activity before and after the addition of each concentration of the Aβ peptide, in order to have an internal control of the activity in the absence of the Aβ peptide to cancel putative minor differences in Cb5R pipetting.The volume of Aβ peptide solution added ranged between 1 and 2.5 µL, pipetted from a freshly prepared solution of a prefixed concentration by dilution of aliquots of the stock solution in the assay buffer.The FAD fluorescence of human recombinant Cb5R with a His 6 -tag (32 nM) has been measured at 25 • C with a Perkin Elmer 650-40 spectrofluorometer in quartz cuvettes of 1 cm pathlength with excitation and emission wavelengths of 460 nm and 520 nm, respectively, and excitation and emission slits of 10 nm.The assay buffer was 20 mM phosphate/0.1 mM diethylenetriaminepentaacetic acid (pH 7).We noticed that the FAD fluorescence of the recombinant enzyme displays a small but steady increase as a function of time.This indicates a weak instability of the recombinant Cb5R in these experimental conditions because its denaturation produces around a 1000-fold increase in the FAD fluorescence [67].Due to this, the operational protocol for data acquisition was as follows: after recording the drift of the fluorescence intensity of the Cb5R in the assay buffer for 5 min, Aβ peptides were added at the concentrations indicated in the figures and the fluorescence intensity was recorded for another 5 min.The increase in fluorescence produced by each Aβ peptide concentration shown in the figures was corrected by the drift of fluorescence intensity drift recorded before the addition of the Aβ peptide.
The steady-state anisotropy of fluorescence, r s , has been calculated from the polarization of fluorescence (P) using the equation [73]: r s = 2 × P/(3 − P). (1) Polarization of fluorescence, P, was calculated using the equation: where I Ş and I T are the fluorescence intensities measured with parallel (0 • /0 • ) and perpendicularly (0 • /90 • ) oriented polarizers, respectively, and G is the correction factor for polarization characteristics of the emission monochromator [62,63,73].A value of G of 1.04 ± 0.02 has been obtained for our fluorimeter and used in our calculations.
The distance (d) between the Cb5R prosthetic group FAD and the dye HiLyte TM -Fluor555 covalently bound to Aβ(1-42), which form a donor-acceptor FRET pair, has been calculated for the cases of 1 and 2 acceptors per donor.In the case of multiple acceptors per donor, the rate constant of total FRET (k T ) is the sum of the rate constant of FRET between each donor-acceptor pair (k i ) that can be formed in the assembly [74][75][76][77].The total FRET efficiency (E) is the sum of the FRET efficiency for each donor-acceptor pair that is possible with the geometrical constraints of the system [74][75][76].For each donor-acceptor pair, we have used the equation for FRET: E = d −6 /(R 0 −6 + d −6 ) [62].In this equation, E is the FRET efficiency and R 0 is the distance for 50% FRET efficiency between FAD and HiLyte TM -Fluor555.The value of R 0 has been calculated in this work using the operational protocol followed from previous works for other FRET donor-acceptor pairs [61][62][63].Briefly, we have applied the equation [62]: R 0 = 9.7 × 10 with the assumption of random orientation between donor and acceptor (K 2 = 2/3) and the relative refraction index of an aqueous medium (n = 1.33).The quantum yield of the donor (Φ D ) and the overlap integral, J(λ), have been calculated as indicated in Section 2 of this article.

In Silico Docking Simulation between Aβ(1-42) and Cb5R
Docking between Aβ(1-42) and Cb5R has been performed using the ClusPro server with the following PDB files: Aβ(1-42) (PDB ID: 1IYT) and Cb5R (PDB ID: 1UMK), access date 29 January 2019.The images and analysis of the model structures were built up with the UCSF Chimera software.

Statistical Analysis
All the results are means ± standard error of the mean (S.E.M.) of experiments performed, at least, in triplicate.
confirm the saturation of the maximum fluorescence change at a concentration of 0.1 µM of His6.The small value of the maximum fluorescence change indicates that His6 interaction with Aβ(1-42) elicits only a small change in the microenvironment of the HiLyte TM -Fluor555 dye, which is bound to ASP1 of Aβ(1-42).The short initial lag phase suggests that conformational changes in His6 and/or Aβ(1-42) HiLyte TM -Fluor555 are needed for a better coupling between both molecules.The dependence upon the His6 concentration of the increase in fluorescence intensity at 2000 s of the kinetic of the increase in fluorescence is shown in Figure 1B.Taking into account that the noise of the fluorescence signal introduces an error close to ±2% in the differences between fluorescence intensity readings, the 50% of the maximum fluorescence change is reached with a concentration of ≈ 50-60 nM of His6, i.e., the saturation of the fluorescence change is observed at ratios ≥10 molecules of His6 per Aβ(1-42) HiLyte TM -Fluor555 monomer.

Figure 1 .
Figure 1.The fluorescence of Aβ(1-42) HiLyte TM -Fluor555 monitors its interaction with His6.(A) Kinetics of the increase in Aβ(1-42) HiLyte TM -Fluor555 fluorescence induced by the addition of His6.For a more direct evaluation of the magnitude of the increase in the fluorescence, the results are presented as the ratio between the fluorescence intensity at different times (F) and the fluorescence intensity at time 0 (F0).The kinetic traces shown are the means of triplicate experiments performed with 10 nM Aβ(1-42) HiLyte TM -Fluor555 and the concentrations of His6 indicated in the figure,

Figure 1 .
Figure 1.The fluorescence of Aβ(1-42) HiLyte TM -Fluor555 monitors its interaction with His 6 .(A) Kinetics of the increase in Aβ(1-42) HiLyte TM -Fluor555 fluorescence induced by the addition of His 6 .For a more direct evaluation of the magnitude of the increase in the fluorescence, the results are presented as the ratio between the fluorescence intensity at different times (F) and the fluorescence intensity at time 0 (F 0 ).The kinetic traces shown are the means of triplicate experiments performed with 10 nM Aβ(1-42) HiLyte TM -Fluor555 and the concentrations of His 6 indicated in the figure, which was added at time 0. The kinetic trace in gray color is the control with the addition of only the buffer in which His 6 is prepared (0 His 6 ).(B) Dependence upon the His 6 concentration of the increase in the fluorescence intensity of Aβ(1-42) HiLyte TM -Fluor555 at the end of the kinetic of increase in fluorescence.Other experimental conditions used for these fluorescence measurements are given in Section 4.

Figure 2 .
Figure 2. Clusters of the structures generated using the MDockPeP Server for the complex between 1His6 and the structure of Aβ(1-42) registered with PDB ID: 1IYT in UniPro Databank analyzed with PDBePISA.Aβ(1-42) backbone is shown in brown and 1His6 peptide is shown in red for the representative models with the lowest estimated binding energy of cluster1, cluster 2 and cluster 3, model 7, model 9 and model 3, respectively.H-bonds between amino acids of both structures are shown as a dotted black line.

Figure 2 .
Figure 2. Clusters of the structures generated using the MDockPeP Server for the complex between 1His 6 and the structure of Aβ(1-42) registered with PDB ID: 1IYT in UniPro Databank analyzed with PDBePISA.Aβ(1-42) backbone is shown in brown and 1His 6 peptide is shown in red for the representative models with the lowest estimated binding energy of cluster1, cluster 2 and cluster 3, model 7, model 9 and model 3, respectively.H-bonds between amino acids of both structures are shown as a dotted black line.
Inhibit the Reduction in Cb 5 Catalyzed by Purified Recombinant His 6 -Tagged Cb5R, and No Inhibition Is Observed after Deletion of the His 6 -Tag

Figure 3 .
Figure 3.The peptide His6 antagonizes the interaction of Aβ(1-42) with calbindin-D28k and CaM.(A) Effect of increasing concentrations of His6 on the kinetics at an increase of 10 nM Aβ(1-42) Hi-Lyte TM -Fluor555 fluorescence after adding 5 nM calbindin-D28k at time 0. (B) Effect of increasing concentrations of His6 on the kinetics at an increase of 10 nM Aβ(1-42) HiLyte TM -Fluor555 fluorescence after adding 5 nM CaM at time 0. The results are presented as the ratio between the fluorescence intensity at different times (F) and the fluorescence intensity at time 0 (F0), and the kinetic traces shown in this figure are the means of experiments performed in triplicate.The color code used for the assayed concentrations of His6 is given in the inset of the figures.Other experimental conditions used for these fluorescence measurements are given in the Materials and Methods section.
Figure 4A highlight the potent inhibition of this activity by nanomolar Aβ(1-42) concentrations.As up to 2 µM of Aβ(1-42), the highest concentration tested in this work, we have

Figure 3 .
Figure 3.The peptide His 6 antagonizes the interaction of Aβ(1-42) with calbindin-D28k and CaM.(A) Effect of increasing concentrations of His 6 on the kinetics at an increase of 10 nM Aβ(1-42) HiLyte TM -Fluor555 fluorescence after adding 5 nM calbindin-D28k at time 0. (B) Effect of increasing concentrations of His 6 on the kinetics at an increase of 10 nM Aβ(1-42) HiLyte TM -Fluor555 fluorescence after adding 5 nM CaM at time 0. The results are presented as the ratio between the fluorescence intensity at different times (F) and the fluorescence intensity at time 0 (F 0 ), and the kinetic traces shown in this figure are the means of experiments performed in triplicate.The color code used for the assayed concentrations of His 6 is given in the inset of the figures.Other experimental conditions used for these fluorescence measurements are given in Section 4.

Figure 4 .
Figure 4. Aβ(1-42) and Aβ(25-35) inhibit the NADH-dependent Cb5 reductase activity of the human recombinant Cb5R with a His6-tag.(A) Kinetics of Cb5 reduction by the human recombinant Cb5R with a His6-tag in the absence and presence of 50 and 100 nM Aβ(1-42).(B) Dependence upon the concentration of Aβ(1-42), Aβ(25-35) and scrambled Aβ(1-42) of the inhibition of the Cb5 reductase activity of the human recombinant Cb5R with a His6-tag.The results shown are the means ± standard error of the mean (S.E.M.) of experiments performed in triplicate.Other experimental conditions used for these assays are given in the Section 4.7.The results of the dependence upon the concentration of Aβ(1-42) and Aβ(25-35) of the inhibition of the NADH-dependent reduction in Cb5 by human recombinant Cb5R with a His6-tag are shown in Figure 4B.These results showed that Aβ(1-42) and Aβ(25- 35)  are equally potent as inhibitors of this activity, without statistically significant differences between both datasets.The inhibitory effect is specific of neurotoxic Aβ peptides, because a peptide with a random sequence of the 42 amino acids of Aβ (scrambled Aβ), which has been found non-toxic in previous works[27,30], does not inhibit this activity (Figure4B).The results of inhibition by Aβ(1-42) and Aβ(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) fit well to the equation for one inhibitor binding site, with an inhibition constant (Ki) between 50 and 60 nM of Aβ monomers, and maximum inhibition (Imax) between 75 and 80%.Of note, this Ki value is nearly 10-fold higher than the Cb5R concentration used in these assays, and, therefore, the contribution of the peptide bound to Cb5R to the total Aβ concentration is lower than 10% at half of the saturation of the inhibition curve.Combining these kinetic results, it merges the conclusion that the Cb5 reductase activity of the His6-tagged Cb5R is strongly sensitized to neurotoxic Aβ peptides.

Figure 4 .
Figure 4. Aβ(1-42) and Aβ(25-35) inhibit the NADH-dependent Cb 5 reductase activity of the human recombinant Cb5R with a His 6 -tag.(A) Kinetics of Cb 5 reduction by the human recombinant Cb5R with a His 6 -tag in the absence and presence of 50 and 100 nM Aβ(1-42).(B) Dependence upon the concentration of Aβ(1-42), Aβ(25-35) and scrambled Aβ(1-42) of the inhibition of the Cb 5 reductase activity of the human recombinant Cb5R with a His 6 -tag.The results shown are the means ± standard error of the mean (S.E.M.) of experiments performed in triplicate.Other experimental conditions used for these assays are given in the Section 4.7.

Figure 5 .
Figure 5. Dependence upon the concentration of Aβ(1-42) and Aβ(25-35) of the increase in FAD fluorescence (ΔF/F0) bound to the human recombinant Cb5R with a His6-tag.There was not a significant difference between the titration of FAD fluorescence with Aβ(1-42) in the absence (filled black circles) and the presence of 5 µM Cb5 (blue triangles).The lines are the results obtained via non-linear regression fit to the equation for 1:1 ligand binding site per protein molecule.The fit of the data yielded the following results: Y = −0.0095+ [0.5227 × x/(96.92+ x)] (R 2 = 0.9870) for Aβ(1-42), and Y = −0.0019+ [0.5311 × x/(99.253+ x)] (R 2 = 0.9974) for Aβ(25-35).The results shown are the means ± S.E.M. of experiments performed in triplicate.Other experimental conditions used for these fluorescence measurements are given in the Materials and Methods section.

Figure 5 .
Figure 5. Dependence upon the concentration of Aβ(1-42) and Aβ(25-35) of the increase in FAD fluorescence (∆F/F0) bound to the human recombinant Cb5R with a His 6 -tag.There was not a significant difference between the titration of FAD fluorescence with Aβ(1-42) in the absence (filled black circles) and the presence of 5 µM Cb 5 (blue triangles).The lines are the results obtained via non-linear regression fit to the equation for 1:1 ligand binding site per protein molecule.The fit of the data yielded the following results: Y = −0.0095+ [0.5227 × x/(96.92+ x)] (R 2 = 0.9870) for Aβ(1-42), and Y = −0.0019+ [0.5311 × x/(99.253+ x)] (R 2 = 0.9974) for Aβ(25-35).The results shown are the means ± S.E.M. of experiments performed in triplicate.Other experimental conditions used for these fluorescence measurements are given in Section 4.

Figure 6 .
Figure 6.FRET between FAD bound to the human recombinant Cb5R with a His6-tag and Aβ(1-42) HiLyte TM -Fluor555.(A) Emission spectrum of the human recombinant Cb5R with a His6-tag with an

Figure 7 .
Figure 7. Simulation generated using docking between the soluble human erythrocytes isoform of Cb5R (PDB ID: 1UMK) and Aβ(1-42) (PDB ID: 1IYT) using the ClusPro Web Server.The polypeptide backbone of Cb5R is shown in gray/green, with the position of the prosthetic group FAD in blue, and the Aβ(1-42) is shown in red.The NH2-terminus amino acid of the Cb5R is labeled with a yellow circle.

Figure 7 .
Figure 7. Simulation generated using docking between the soluble human erythrocytes isoform of Cb5R (PDB ID: 1UMK) and Aβ(1-42) (PDB ID: 1IYT) using the ClusPro Web Server.The polypeptide backbone of Cb5R is shown in gray/green, with the position of the prosthetic group FAD in blue, and the Aβ(1-42) is shown in red.The NH 2 -terminus amino acid of the Cb5R is labeled with a yellow circle.

Molecules 2023 , 22 Figure 8 .
Figure 8. SDS-PAGE of human recombinant Cb5R.Lane 1: His6-tagged Cb5R.Lane 2: Cb5R after the removal of His6 by treatment with the Thrombin Clean Cleavage TM kit and passage through a Sephadex G75 column.MWM, lane of molecular weight markers.The gel showed that the treatment with the Thrombin Clean Cleavage TM kit produced the expected decrease of ~1 kDa in the molecular weight and no further proteolytic degradation of Cb5R.

Figure 8 .
Figure 8. SDS-PAGE of human recombinant Cb5R.Lane 1: His 6 -tagged Cb5R.Lane 2: Cb5R after the removal of His 6 by treatment with the Thrombin Clean Cleavage TM kit and passage through a Sephadex G75 column.MWM, lane of molecular weight markers.The gel showed that the treatment with the Thrombin Clean Cleavage TM kit produced the expected decrease of ~1 kDa in the molecular weight and no further proteolytic degradation of Cb5R. .

Table 1 .
Interacting interfaces for the complex formation between His 6 and Aβ peptides obtained via molecular docking simulation.