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• W2068039154 abstract "TTR and TTR bind by x-ray crystallography (View interaction) Protein misfolding and aggregation are involved in the pathogenesis of particularly important human diseases, the amyloidoses. Such diseases are characterized by the extracellular deposition of one of more than 30 amyloidogenic proteins as cross-β-sheet amyloid fibrils [1, 2]. Specific mutations may induce or enhance the amyloidogenic potential of several amyloidogenic proteins, among which transthyretin (TTR) represents a notable example. Most mutations in TTR are involved in familial amyloidotic polyneuropathy (FAP) and cardiomyopathy (FAC), which are related to the predominant deposition of fibrillar aggregates of TTR in peripheral nerves and the heart, respectively [3]. TTR is a homotetramer of about 55 kDa involved in the transport of thyroxine (T4) in extracellular fluids, both plasma and cerebrospinal fluid. The assembly of the four identical subunits in TTR is highly symmetrical, being characterized by 222 symmetry. Each monomer is composed of eight antiparallel β-strands, arranged in a topology similar to that of the Greek key β-barrel, and a short α-helix. Two monomers are held together to form a stable dimer, and two dimers associate back to back, mainly through a limited number of hydrophobic contacts, to form the tetramer. A long channel, at the interface between the two dimers and coincident with one of the two-fold symmetry axes, transverses the tetrameric protein. Each symmetric half of the channel harbors a funnel-shaped binding site for T4. A large body of evidence has been obtained to indicate that the rate-limiting dissociation of the native tetrameric state into monomers, followed by misfolding of TTR monomers and their downhill polymerization, is required to generate protein aggregates in vitro and presumably in vivo [4]. Following these observations, the properties of a large number of compounds have been investigated in prospect of their use as drugs effective in the therapy of TTR amyloidoses [4]. A key feature they must possess is their ability to fit into the TTR T4 binding sites, establishing interactions with residues of the couple of subunits that line each hormone binding cavity present in the central channel. As a result, such interactions would bridge neighboring subunits at the dimer–dimer interface, thereby stabilizing the TTR tetramer. Indeed, one such compound, tafamidis, was found to possess quite favorable features in vitro and in vivo, such as a highly selective binding to TTR in human plasma, and was recently approved by EMA (European Medicines Agency) for the treatment of TTR FAP [5]. The crystal structures of amyloidogenic TTR variants are generally well conserved under native conditions [6]. On the other hand, structural alterations in the crystal structures of some amyloidogenic TTR variants crystallized at moderately acidic pH (pH 4.6) [7, 8] and of wt-TTR crystallized at pH 4.0 [9] have been revealed. It should be noted that an acidic pH is also required to promote in vitro TTR fibrillogenesis, which is mediated by protein destabilization occurring at this pH [10]. Most evident is the alteration affecting I84S TTR, in which the lowering of the pH (pH 4.6) causes the unwinding of the TTR short EF-helix (residues 75–82) and the change in conformation of the adjacent EF-loop hosting the mutation (residues 83–90) in one (B subunit) of the two subunits (A and B subunits) present in the asymmetric unit [7]. Through the use of X-ray analyses we show here that the interactions of two chemically distinct fibrillogenesis inhibitors with I84S TTR prevent the acidic pH-induced conformational change in this amyloidogenic variant. Recombinant wild type (wt-) and I84S human TTR were prepared and quantified as described [7]. trans-resveratrol, T4 and diflunisal were from Sigma–Aldrich. CHF5074 was synthesized according to [11]. Oil B was purchased from Hampton Research (CA). All chemicals were of analytical grade. To highlight the inhibitory effect of diflunisal and CHF5074 on in vitro TTR fibrillogenesis at moderately acidic pH, kinetics of fibril formation were monitored by following the increase in turbidity, estimated spectrophotometrically at 400 nm, upon incubation of I84S TTR or wt-TTR, at final concentrations of 3.6 μM, with either CHF5074 or diflunisal, at a final concentration of 10.5 μM. According to the procedure adopted for other fibrillogenesis inhibitors [12], TTR was incubated with fibrillogenesis inhibitors at neutral pH (10 mM Na phosphate buffer, pH 7, 100 mM KCl, 1 mM EDTA) for 3 h at room temperature, prior to incubation of the protein at acidic pH upon addition of an equal volume of 100 mM sodium acetate, 100 mM KCl, 1 mM EDTA, pH 4.2 (final pH 4.3), at 37° C, in order to promote fibrillogenesis. Crystals of ligand–TTR complexes were obtained at room temperature in about one week by cocrystallization, using the hanging-drop vapor diffusion method. Crystals of I84S TTR in complex with either CHF5074 or diflunisal at pH 4.6 were obtained as described previously for uncomplexed I84S TTR at acidic pH [7]: hanging drops containing the TTR variant (5 mg/ml) and a fourfold molar excess of each ligand were equilibrated against 15% (w/v) PEG 2000 monomethylether, 0.1 M ammonium sulfate, 0.05 M sodium acetate buffer (pH 4.6). Small aliquots of concentrated ligand stock solutions in DMSO were added in cocrystallization experiments (final DMSO concentration in the crystal medium upon equilibration of approx. 4% (v/v)). In a control experiment 4% (v/v) DMSO was not found to affect significantly the fibrillogenesis kinetics for I84S TTR at acidic pH (Supplemental Fig. S6). To obtain crystals of wt-TTR in complex with CHF5074 at neutral pH, hanging drops containing wt-TTR (7 mg/ml) and a fourfold molar excess of CHF5074 were equilibrated against 2 M ammonium sulfate, 0.1 M KCl, 0.05 M sodium phosphate pH 7.0. The datasets concerning the different ligand–TTR complexes were collected at the synchrotron radiation facility ESRF in Grenoble, using beamlines ID23-2 and ID14-eh1. All crystals were briefly soaked in oil B as a cryoprotectant before freezing and data collection. Datasets were processed with the software Mosflm [13] and scaled with Scala [14] contained in the CCP4 suite [15]. The structures of I84S TTR–ligand complexes were refined starting from that of the dimer of wt-TTR as a template (Protein Data Bank (PDB): 1F41, [6]). The models were subjected to rigid-body minimization and subsequently to refinement steps with Phenix [16] or Refmac [17]. Map visualization and manual adjustment of the models were performed using the Coot graphic interface [18]. Atomic coordinates of the inhibitor molecules and restraints were obtained through the PRODRG server [19]. Two fibrillogenesis inhibitors have been cocrystallized with TTR: diflunisal, a previously characterized fibrillogenesis inhibitor which is currently being tested in human clinical trials in FAP patients [20], and CHF5074, a chlorinated derivative of flurbiprofen [11] (Fig. 1 a, Table 1 ). Fluorometric competitive binding assays, using resveratrol as fluorescent probe, have shown that CHF5074 possesses high binding affinity for both wt-TTR and I84S TTR (Supplementary data, Figs. S1–S4). Effective inhibitions of fibril formation in the presence of saturating CHF5074 and diflunisal at moderately acidic pH have been found for both I84S TTR and wt-TTR (Fig. 1b and Supplemental Fig. S5, respectively), consistent with previously reported fibrillogenesis inhibitions of I84S TTR and wt-TTR by fibrillogenesis inhibitors [12]. In the crystal structures of TTR–CHF5074 complexes the TTR-bound ligand is relatively well defined for both T4 binding sites in the TTR tetramer. The inhibitor is bound identically, in the “forward mode”, in both wt-TTR cocrystallized at pH 7.0 and I84S TTR cocrystallized at pH 4.6: the di-chloro phenyl ring is deeply buried inside the central channel, and the carboxylic moiety protrudes towards the solvent (Fig. 1c). Its electron density, shown in Fig. 2 a and Supplemental Fig. S7 for CHF5074 in complex with wt-TTR at pH 7,0 and I84S TTR at pH 4.6, respectively, was clearly visible from the first stages of refinement. It is well defined for inner and medium rings, whilst the ligand portion comprising the cyclopropane ring and the carboxylate end group is poorly defined, suggesting a rather loose interaction for this ligand moiety (Figs. 2a and S7). It should be noted, however, that, as in all the other cases of binding of ligands to the central channel of human TTR, the molecular (and crystallographic) symmetry of the channel implies the presence of two possible orientations for the ligand within each binding site, giving rise to an intrinsic disorder in its electron density. The funnel-shaped T4 binding site is characterized by three subsites, each composed of pairs of symmetric halogen binding pockets (HBPs), wherein the iodine atoms of bound T4 are accommodated: an outer binding subsite (HBP 1 and 1′), an inner binding subsite (HBP 3 and 3′), and an intervening interface (HBP 2 and 2′), respectively formed by residues Lys15, Leu17, Thr106, and Val121, residues Ser117, Leu110, Thr119 and Ala108, and residues Leu17, Ala108, Ala109, Leu110, including the methylene carbons of Lys15 (PDB: 1ROX, [21]). Both TTR-bound CHF5074 molecules make hydrophobic and electrostatic interactions with residues lining the TTR binding cavities. The latter are represented by four polar contacts (Fig. 1c): the carboxylate of CHF5074 is at a close distance to Lys15 ε-NH3 + (2.48–3.15 Å); the para chlorine atom of the di-chloro phenyl ring is close to the –OH group of Ser117 (Clδ–Oγ 3.8 Å); the meta chlorine atom interacts with atoms of the Ser117–Thr118 peptide bond (Clε–O117, 3.36 Å; Clε–N118, 3.71 Å); the fluorine atom interacts with the carbonyl oxygen of Ala109 (F–O109, 3.52 Å). The Lys15 side chain is held in place by Glu54 (Oε2–Nζ 2.79 Å), which in turn interacts with His56 (Nε2–Oε1 2.67 Å). The Ser117 side chain is the only one that presents a different conformation as compared to the apo-protein: its –OH group is in fact rotated around the Cβ–OH bond. Notably, in this new situation Ser117 and Ser117′ of B and B′ subunits participate in H-bond interactions with the two Ser117 residues present in A and A′ subunits (Fig. 1c). The latter interactions between subunits may thus provide an additional contribution to the stabilization of tetrameric TTR. Residues making hydrophobic contacts with the ligand are Leu17 and Leu17′, Ala108 and Ala108′, Leu110, and Leu110′, Thr119 and Thr119′. A comparison of our structure with that of TTR in complex with flurbiprofen (Fig. 1 a) (PDB: 1DVT, [22]) indicates that the most relevant difference between the interactions of the two ligands with TTR is due to the presence of the two chlorine atoms in CHF5074: they better fill the HBP-3 and HBP-3′ regions, participating in polar interactions. The situation for I84S TTR in complex with diflunisal at acidic pH is different. Here, the electron density is less defined, suggesting the presence of two orientations (forward and reverse) of the ligand, in addition to the twofold symmetry, as the cause of disorder in the electron density (see PDB: 3D2T [23] for the structure of wt-TTR in complex with diflunisal, which also reveals different orientations for the TTR-bound inhibitor). The Fourier-difference electron density map for diflunisal in complex with I84S TTR at acidic pH is shown in Supplemental Fig. S8. We have previously shown that I84S TTR is a conformationally labile genetic variant, which undergoes a pH-dependent significant conformational change, characterized by the loss at acidic pH of the EF-helix and the alteration of the adjacent EF-loop in only one of the two subunits present in the asymmetric unit [7] (Fig. 2b and c). It should be pointed out that a large conformational change affecting the same protein region (residues 75–90) in subunits B was also observed for wt-TTR crystallized at pH 4.0 [9]. However, in this case the EF-helix slips away from its original position, at variance with the unfolding of the helix in the case of I84S TTR at pH 4.6. Moreover, a perturbation of the same region was observed for tetrameric TTR, formed by the association of F87/L110 TTR monomers, in complex with Zn2+ [24]. Regarding the possible cause of the asymmetry of the conformational change in the tetrameric protein, affecting only half of the four subunits [7, 9], it should be noted that residue I84 is contiguous with the short α-helix (residues 75–82) and is located in the region involved in intermolecular contacts in the crystal lattice. While at neutral pH the four α-helices of I84S TTR are stable enough to be compatible with their maintenance as in the case of wt-TTR [7], at pH 4.6 a destabilization of the helices takes place, so that intermolecular contacts are also affected. In particular, a new situation selected by crystal lattice would permit the interaction of an intact α-helix of a subunit of a dimer (the asymmetric unit) with a loop replacing the α-helix of a subunit of the symmetry related dimer (PDB: 2G3X, [7]). On the basis of this hypothesis, one can assume that in solution, in the absence of constraints imposed by crystal lattice, the pH-induced conformational change likely affects all subunits of the I84S TTR tetramer. The main aim of this work was to establish whether the presence of two distinct fibrillogenesis inhibitors, CHF5074 and diflunisal, bound in the T4 binding pockets of TTR could stabilize the native structure of I84S TTR at acidic pH, permitting the maintenance at low pH of intact intermolecular contacts as for wt-TTR or I84S TTR at neutral pH. Indeed, the crystal structures of I84S TTR in complex with CHF5074 or diflunisal are very well conserved, even at acidic pH: the superimposition of the structure of wt-TTR in complex at neutral pH with CHF5074 on the structure of the I84S TTR variant in complex with CHF5074 or diflunisal at acidic pH (pH 4.6) gives r.m.s.d. between equivalent Cα atoms of 0.34 and 0.29 Å, respectively. The comparison with the structure of uncomplexed wt-TTR (PDB: 3CFM, [25]) gives a very similar value, 0.38 Å, whilst the same comparison between the uncomplexed I84S structure at low pH and the structure of I84S TTR in complex with CHF5074 at low pH gives values of 0.49 Å for monomers A and 2.12 Å for monomers B. Therefore, it can be inferred that the bound fibrillogenesis inhibitors (either CHF5074 or diflunisal) confer a remarkable stability to the I84S TTR tetramer, as they are able to readjust the altered structure of uncomplexed I84S TTR at acidic pH, converting it to that typical of wt-TTR at neutral pH. The analysis of the thermal parameters of the main chain atoms, illustrated in Fig. 3 , for I84S TTR at pH 4.6, either as uncomplexed protein or in complex with CHF5074, and for wt-TTR at pH 7.0, has also been performed. In all cases the core of the A–B dimer is quite rigid in the crystal, whilst most loops are rather flexible, as expected. In particular, the relatively most flexible parts are loops 36–42 of monomer A and 98–103 of monomer B. The EF-helix and the adjacent long loop that includes residue 84 are also rigid or relatively rigid for both monomers A and B in the case of wt-TTR at pH 7 and of the CHF5074-I84S TTR complex at pH 4.6, whilst in uncomplexed I84S-TTR at pH 4.6 the latter area becomes very flexible in monomer B. The finding that a large protein region (residues 75–90) undergoes a remarkable conformational change in both I84S TTR at pH 4.6 [7] and wt-TTR at pH 4.0 [9] in two subunits of the TTR tetramer indicates that it is rather flexible within an otherwise rigid scaffold. It has been hypothesized that the observed changes in such region likely destabilize the tetrameric protein and may represent initial events occurring in the amyloidogenic process [7, 9]. In all tested cases, the structure of TTR in complex with fibrillogenesis inhibitors remains nearly unchanged at both neutral and acidic pH, indicating that the ligand stabilizes the native protein structure mainly by filling the empty central cavity, without modifying the protein conformation. In particular, the remarkable conformational change we have observed at pH 4.6 in subunit B for I84S TTR [7], is hampered by the presence of the ligand. TTR-bound CHF5074 or diflunisal does not establish any specific interaction with residues of the area involved in the conformational change: the shortest distance between the Cα atom of residue 84 and the closest atom of the ligand is in fact about 20 Å for both monomers (Fig. 2c). We have to conclude that the effect of the ligand is not direct, but it must be attributed to an overall stabilization of the tetramer itself. In particular, the direct interactions that the two bound ligands establish with residues of the central channel of TTR appear to have a long-range effect resulting in the rigidification of the tetramer, by reducing the flexibility of the entire structure and, consequently, the possibility of transition into the altered conformation induced by a moderately acidic pH in the I84S TTR variant. It should be noted that two chemically distinct fibrillogenesis inhibitors, such as CHF5074 and diflunisal, whose mode of binding to T4 sites of TTR is rather different, exhibit a quite similar stabilizing effect on TTR structure. This seems to suggest a common mechanism of stabilization for fibrillogenesis inhibitors. Overall, this study provides direct evidence, on a structural basis, for the ability of specific ligands of TTR to preserve the native structure of this amyloidogenic protein. Atomic coordinates and structure factors have been deposited in the PDB. PDB ID codes are 4I85, 4I87 and 4I89, respectively for wt-TTR–CHF5074 complex (pH 7.0), I84S TTR–CHF5074 complex (pH 4.6) and I84S TTR–diflunisal complex (pH 4.6). We thank the staff of beamlines ID23-2 and ID14-1 of European Synchrotron Radiation Facility, Grenoble (France), for technical assistance during data collection. The technical assistance of Claudia Rasore is also gratefully acknowledged. This work received financial support from MIUR (PRIN Project 2009KN2FBM), Rome, Italy, from Chiesi Farmaceutici, Parma, Italy, and from the Universities of Padua and Parma, Italy. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2013.06.016. Supplementary data 1. Supplementary data 2. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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• W2068039154 title "Structural evidence for native state stabilization of a conformationally labile amyloidogenic transthyretin variant by fibrillogenesis inhibitors" @default.
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