Transtyretiini ja Kupari (Cu)

 https://www.nature.com/articles/s41598-018-31808-5

Discussion

TTR functions as a transporter in plasma and in the brain of several molecules, such as T4 and retinol. In the CSF, TTR is also recognized as the main Aβ binder, and, it is able to transport Aβ across the brain-blood barrier in the brain-to-blood direction⁸. The details of this clearance mechanism, however, remain to be elucidated.

The concentration of TTR is different in the blood and in the CSF: 3–4 μM and 0.1–0.4 μM, respectively²⁴. Under physiological conditions, the soluble Aβ peptide is secreted into the synaptic cleft of normal subjects and AD patients, and its concentration is in the nanomolar range⁴⁰. In the brain, the concentration of Aβ is about six-times higher than in plasma. Perturbations in the efflux of Aβ out of the brain could affect the levels of soluble Aβ in the CSF^12,41. Moreover, when metals ions are released into the synaptic space during neurotransmission, they can modify the stability of the Aβ peptide. In the AD affected brain, the total concentration of Cu and other metals contained in amyloid plaque deposits has been established at 400 μM²⁶.

In this work, we have investigated the interaction between TTR and Aβ by using a segment (residues 1–28) of the full length Aβ peptide as it is more soluble and stable in buffer solutions. More importantly, however, Aβ(1–28) contains the hydrophobic core VFF (residues 18–20), which is recognized by TTR^7,23. Furthermore, Aβ(1–28) holds the residues that chelate metal ions³¹ and that can mediate the interaction between TTR and Aβ. It is not known if TTR binds the entire sequence of Aβ or only the fragments of Aβ cleaved by TTR^6,42, but the segment 1–28 of Aβ is exposed in amyloid fibrils of Aβ(1–42)⁴³.

Our BLI study has revealed that the affinity of TTR for Aβ(1–28) is modulated by copper. The binding of TTR to immobilized Aβ(1–28) increases with CuCl₂ concentration from 0 to 12.5 mM (Fig. 1). When CuCl₂ is included in the BLI dissociation buffer, no dissociation is observed Fig. 1C, consistent with the hypothesis that Cu²⁺ plays a key role in the stability of the TTR-Cu-Aβ complex.

Under pathological conditions, the pH dramatically decreases in the CNS, in plasma and in cells^44,45,46. It is known that copper binding to histidines enhances Aβ aggregation at the pH typical of physiological acidosis⁴⁷. The pH dependence of this interaction supports the involvement of the histidine side-chains. Consequently, the majority of our BLI and crystallographic experiments were done at acid pH (pH 5.5). Indeed, acidic pH and elevated CuCl₂ concentration in the crystal soaking experiments (Table 1) favors a change in the TTR conformation in the stretch of residues 72–92 (see Supplementary Fig. S3).

The metal ion concentration needed to induce the conformational change in TTR crystals is necessarily elevated compared to the conditions in solution as the system is constrained by lattice packing forces. When TTR crystals of the P2₁2₁2 crystal form are soaked in FeCl₂ at acidic pH, only the B monomer in the asymmetric unit changes its conformation, whereby the E-F helix and the stretch of residues 85–92 undergo a rearrangement that is accompanied by a variation in the dimer-dimer interface. A similar change in conformation was observed when P2₁2₁2 TTR crystals were soaked with a Re-complex³⁰. Although the binding of various metals provokes similar changes, the changes induced differ in their extent. Three key distances can be used to evaluate the magnitude of the conformational change (see Supplementary Table S3): the separation between the four Ile-110 residues in the tetramer (to monitor the rotation of the dimers relative to each other), the distance between Gly-83B on the E-F loop from Thr-123A (to measure the shift of the E-F helix), and the separation between two Asp-38 residues (to assess the relationship between the monomers that form the dimer). This last measure provides a distinctive signature of metal binding. The Asp-38–Asp-38 separation, typically 21.5 Å in the absence of metals, shortens to 20.3 Å in the TTR-Mn complex and to 17.8 Å for the TTR-Zn complex (PDB code: 3DGD²⁸), while in the TTR-Cu complex it increases to 25.7 Å and 26.3 Å in the TTR-Re complex (PDB code: 5K1N³⁰) up to a maximum of 27.0 Å for the TTR-Fe complex (see Supplementary Table S3).

Metal chelation by proteins often involves the repositioning of certain residues. When considering TTR-Aβ complexes it is important to take into account that both partners in the interaction may alter their conformation in a dynamic and polymorphic manner. Fe promotes Aβ aggregation⁴⁸ that interferes with the dynamics of amyloid formation⁴⁹, and its metal binding is pH-dependent and affects oligomerization⁵⁰. Morphological differences may affect toxicity. Aβ aggregates containing Zn, Fe or Cu are neurotoxic^51,52.

Studies in vitro are focused on Aβ interactions with Cu and Zn, even if Mn also binds Aβ peptides³¹ and plays a role in neurodegeneration. In order to investigate the importance of Mn in the TTR-Aβ interactions, TTR crystals were soaked in 5 mM of MnCl₂ at pH 6.0. No significant conformational changes were observed, suggesting that even if Mn binds Aβ³¹, it does not play a relevant role in the interactions between TTR and Aβ.

Although Cu²⁺ induces a conformational change in TTR (Fig. 3; X-ray anomalous differences validate the presence Cu sites see Supplementary Table S2) which resembles those revealed by the Fe²⁺ and Re²⁺ experiments, the manner in which Cu²⁺ is chelated differs from those observed for Fe²⁺ or Re (Fig. 2). Even if both His-88 and His-90 are involved in Cu and Zn chelation, the interaction from the third residue is different, being Asp-74 for Cu²⁺ (Fig. 3) and Glu-92 in the case of Zn²⁺ at pH 4.6²⁸. The network of residues that intervene in metal binding is quite extensive (see Supplementary Fig. S3), and most residues have been recognized as possible zinc binders. The mutation of His-90 to Ala results in the loss of TTR catalytic activity and its ability to disrupt fibrils⁴².

Despite the fact that the TTR-Aβ interaction can be demonstrated in the presence of Cu by BLI, TTR crystals grown in the presence of CuCl₂ and Aβ did not show any ordered Aβ peptides. This implies that the crystallization forces of TTR molecules disrupt the TTR-Aβ complex. Given the size of the Aβ(1–28) peptide with respect to TTR monomers (127 aa), one would expect the TTR-Aβ complex to crystallize in rather different unit cell constants and/or space group.

However, the TTR crystal structure contains 30Å -wide diamond-shaped solvent channels, which run the length of the crystal, and the likely presence of Aβ in the crystal interstices could be inferred from lower Cu²⁺ binding due to Aβ chelation. Under identical soak conditions, the TTR conformational change is characterized by a smaller Asp-38–Asp-38 separation (23.9 Å) compared to that seen in the absence of Aβ (25.0 Å). An additional Cu binding site is located in proximity of Glu-54 (Fig. 3E,F). This residue has been identified in regards to Cr³⁺ binding, and its involvement in Cu²⁺ binding has been suggested⁵³. Thus, Glu-54 could act as the gatekeeper to the tunnel that leads to the four Leu-110 residues at the center of the tetramer, a residue that when mutated to alanine abolishes TTR’s Aβ scavenging activity. A contact between the Aβ peptide and Leu-110 would require a transit in front of Glu-54.

Our results show that TTR changes its conformation in response to binding Fe^2+ 54 (as opposed to Fe³⁺ as confirmed by XANES experiments, see Supplementary Information Fig. S4) and Cu²⁺ at acidic pH, and that Cu is essential for the recognition of Aβ(1–28) by TTR. Only certain divalent metals ions (Cu²⁺, Fe²⁺) provoke the crystallographic conformational change. When TTR is soaked with trivalent metal ions under acid pH conditions (Al³⁺, Gd³⁺, or Fe³⁺), no conformational changes were observed.

The ability of Cu to promote the formation of the TTR-Aβ complex, and presumably the cerebral clearance of Aβ, is consistent with the report that mice with a defective Cu transporter (which removes Cu) have higher Cu levels, a reduced number of amyloid plaques and diminished plasma Aβ⁵⁵. The lack of binding Aβ by TTR in the absence of Cu, may appear to be in contradiction with experiments carried out with 125I-labeled Aβ (1–40) and Aβ(1–28) peptides by Schwarzman et al., when it was shown that TTR recognizes Aβ in CSF fluid, inhibits its aggregation and prevents fibril formation⁵. However, excluding the possibility that the iodinated peptide used might interact via any of the three TTR halogen binding pockets in the TTR central tunnel, the results may also imply that in the absence of a metal, different polymorphic forms of Aβ are recognized by TTR. By stabilizing a Aβ conformation with mild affinity for TTR, Cu²⁺ ions rescue the interaction. TTR may also bind with higher affinity polymeric Aβ which acquires an alternative and stable conformation through Aβ-Aβ interactions. The immobilization method used here involves Aβ(1–28) mono-biotinylated at the N-terminus (Fig. 1B), and the results are consistent with studies showing that non-aggregating Aβ fragments do not quench TTR tryptophan fluorescence⁵⁶. It is possible that the same peptide, if allowed to dimerize via the LVFFA stretch, may also adopt a conformation that is recognized by TTR in the absence of Cu. The polymorphic diversity of the longer Aβ (1–40/1–42) peptides, with extensive hydrophobic stretches, is likely to generate stable conformations with higher affinities for TTR. Modifying the BLI to function with more complex aggregates may be challenging, but will be necessary to guide the crystallographic structure determination of the TTR-Aβ complex with and without metals.

To conclude, the TTR-Cu conformation provides an alternative starting point for the design of molecules that aim to stabilize a form of TTR with an enhanced Aβ-scavenging activity to counter the reduction of TTR expression in AD patients⁵⁷. As such, these results emphasize Cu as a “ forgotten factor” upon which the TTR-Aβ interaction may depend. Furthermore, metal chelation therapies, which are useful to reduce oxidative stress, should also take into account the essential levels of Cu required for Aβ clearance.