The Crystal Structure of Amyloidogenic Leu55→ Pro Transthyretin Variant Reveals a Possible Pathway for Transthyretin Polymerization into Amyloid Fibrils*

The x-ray crystal structure of the amyloidogenic Leu55 → Pro transthyretin (TTR) variant, implicated as the causative agent in early-onset familial amyloidotic polyneuropathy (Jacobson, D. R., McFarlin, D. E., Kane, I., and Buxbaum, J. N. (1992) Hum. Genet. 89, 353–356), has been solved by molecular replacement, refined at 2.7 Å to a R cryst value of 0.190 (F obs > 2.0ς), and compared with wild-type transthyretin to understand the molecular mechanism(s) involved in amyloidogenesis. Leu55 → Pro TTR crystallizes in space group C2, with eight monomers in the asymmetric unit, and the observed packing contacts are considerably different from those described for the wild-type protein. Refinement of the crystal structure shows that the proline for leucine substitution disrupts the hydrogen bonds between strands D and A, resulting in different interface contacts. Based on the assumption that the observed packing contacts may be significant for amyloidogenesis, a model for the TTR amyloid is proposed. It consists of a tubular structure with inner and outer diameters approximately of 30 and 100 Å and four monomers per cross-section.

Transthyretin (TTR), 1 also known as thyroxin-binding prealbumin, is a plasma homotetrameric protein present in mammals, birds, and reptiles (2). It is synthesized in the liver, choroid plexus of the cerebral ventricles, and retina (3). The protein binds the complex retinol-retinol binding protein, preventing its glomerular filtration (4), thyroxin and related compounds, and also polyhalogenated biphenyl compounds (5). The half-life of TTR is 2-3 days in humans (6), and the major site of TTR degradation is the liver followed by the muscle and skin.
TTR is associated with amyloid deposition in several organs in particular peripheral nerves, cardiac tissue, and ocular vitreous. The majority of the transthyretin-associated amyloidoses are due to single amino acid substitutions, and in senile systemic amyloidosis, the non-mutated protein is present in the amyloid fibrils (7). Familial amyloidotic polyneuropathy is characterized by an autosomal dominant mode of inheritance, with the usual onset of clinical disease appearing about age [25][26][27][28][29][30][31][32][33][34][35]. Peripheral and autonomic neuropathies are prominent and early features of the disease (8).
Several hypotheses have been suggested to explain the amyloidogenic properties of TTR that lead to neurotoxicity and organ dysfunction (9). It has been proposed that there is a conformational state, different from the one presented by the wild-type protein, prior to fibril formation (10). Therefore, x-ray crystallographic comparison studies of the structures of the wild-type and amyloidogenic TTR variants will contribute to explain the amyloidogenic potential of TTR. Defining these structural differences is important, not only in understanding the pathogenesis of the disease, but also in devising therapeutic agents to combat the disease.
Blake et al. (11) reported the crystal structure of the wildtype protein. The protein crystallizes in space group P2 1 2 1 2 with a dimer in the asymmetric unit. The TTR monomer, with 127 amino acid residues (12), contains eight strands, A throughout H, of seven to eight residues in length. The exception is strand D, which is three residues in length. The eight strands form two sheets of four strands each, DAGH and CBEF, arranged in a topology similar to the classic greek key barrel. Two monomers are related by a noncrystallographic 2-fold axis, and they are joined along the FH border to form the dimer. The dimer is composed of two eight-stranded ␤-sheets, DAGHHGAD and CBEFFEBC. The tetramer consists of two dimers related by a crystallographic 2-fold axis. The connecting edges occur between the AB loop of one dimer with the H strand of the other dimer.
The x-ray crystallographic structures of the amyloidogenic variants TTR Met 30 (13,14), TTR Ile 122 (15), and TTR Ser 84 (16) revealed an overall structural homology with the wild-type protein. Only small differences were detected, including a higher spacing between DAGH and CBEF sheets in the TTR Met30 monomer along with a movement of strand A exposing residue 10 to solvent (14). Damas et al. (15) reported an increase in the length of the hydrogen bonds between the Val 122 3 Ile TTR dimers. It was proposed that these changes could lead to the destabilization of the tetrameric structure, thus promoting the formation of an intermediate structure that polymerizes into amyloid fibrils.
Preliminary results, concerning the crystallization procedure of the highly clinically aggressive mutant Leu 55 3 Pro TTR, were reported previously (17). To learn more about the structure/pathogenesis relationship in TTR variants, the structure of amyloidogenic Leu 55 3 Pro TTR variant was refined, compared with wild-type TTR, and is described in the present work.   (17). The Leu 55 3 Pro TTR crystal structure was determined by molecular replacement using the AmoRE software package (18) and the coordinates of one monomer of the wild-type protein (PDB entry 1TTA, Ref. 13). The results from the rotation, translation functions, and the arrangement of the eight monomers in the asymmetric unit were reported previously (17).
There are eight monomers in the asymmetric unit and an initial model with the Leu 55 3 Pro substitution was submitted to restrained least squares minimization refinement using the CCP4 (19) version of PROTIN/PROLSQ programs (20). Least squares refinement, using xray data with maximum resolution 2.7 Å, included the application of non-crystallographic and stereochemical restraints, to increase the data/parameter ratio. The stereochemical restraints were applied by using a library of stereochemical data, which contains a collection of target bond lengths and bond angles established by small molecule studies of 20 amino acids, high resolution studies of small peptide structures, and proteins. The non-crystallographic restraints were not used during the last stages of refinement, to evidence the differences between the eight monomers in the asymmetric unit.
The Fourier syntheses were calculated using the CCP4 software package and were visualized using the graphics software package O (21).
Contact areas involved in dimer-dimer and monomer-monomer interactions were calculated using the CCP4 programs Areaimol and Resarea (19). Coordinates of the refined Leu 55 3 Pro TTR crystal structure and the coordinates of wild-type TTR, deposited in the PDB under entry code 1TTA (13), were used.
Atomic coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank under entry code 5TTR. 55 3 Pro TTR Monomer-Leu 55 3 Pro TTR crystallizes in space group C2, with eight monomers in the asymmetric unit and cell dimensions a ϭ 149.99 Å, b ϭ 78.74 Å, c ϭ 98.95 Å, ␤ ϭ 100.5°.

X-ray Crystal Structure of Leu
Of a total of 127 amino acid residues in the monomer, the final refined model consists of eight monomers of 116 amino acid residues each. The N-and C-terminal residues (1-9 and 126 -127, respectively) are not defined in the electron density maps. Similar disorder was reported for the wild-type TTR (11) and Val 122 3 Ile TTR (15).
The crystallographic R-factor (R cryst ) for the refined Leu 55 3 Pro TTR protein model is 19.9% for all reflections between 8 and 2.7 Å and 18.1% for reflections with F obs Ͼ 3.
The model has good stereochemistry: bond lengths have a root mean square (r.m.s.) deviation of 0.019 and 0.030 Å for angle-bonded distances. The overall temperature factor for the protein main chain atoms varies between 23.7 and 32.3 Å 2 for the eight monomers (Table I).
The Ramachandran plot calculated using PROCHECK (22) showed no residues with main-chain dihedral angles in disallowed regions: 89% of the non-glycine residues are in most favored regions, 10.3% in additional allowed regions, and 0.7% in generously allowed regions. The observed hydrogen-bonding pattern also indicates that the protein model obtained after refinement is correct. Fig. 1 shows the 3F obs Ϫ 2F calc electron density map in the region of the Leu 55 3 Pro mutation.
Comparison of the wild-type and Leu 55 3 Pro mutant TTR (Fig. 2) shows significant deviations between the C␣ positions corresponding to the strand D (residues 54 -56) and loop FG (residues 97-103). Minor deviations are also observed in ␣-helix and loop AB. In fact, in the mutant monomer, residues 54 -56 belong to a long surface loop that connects strands C and E (according to the wild-type TTR nomenclature). This loop (residues 48 -66) is involved in the crystallographic packing as illustrated in Fig. 3.
Thus, the Leu 55 3 Pro monomer structure is organized in seven strands and one ␣-helix, in a topology similar to the classic ␤-barrel.
Intermolecular Interactions in Leu 55 3 Pro TTR Crystal Structure-Leu 55 3 Pro TTR variant crystallizes nonisomorphously with wild-type TTR, unlike other amyloidogenic transthyretin variants, whose structures are described in the literature (13)(14)(15)(16). The crystal asymmetric unit contains four dimers, which assemble into one tetramer in a general position and two dimers near the twofold axes. This non-crystallographic tetramer is formed through hydrogen bond interactions involving the AB loop and strand H, as observed for the wildtype TTR tetramer, although the length of the hydrogen bonds formed is longer (Table II), resulting in an overall less stable tetramer.
Impairment of dimer-dimer contacts, resulting in decreased tetramer stability, was reported previously from biochemical studies (23,24). However, the present work is unique in providing a detailed information about the interatomic contacts between the pathologic Leu 55 3 Pro TTR dimers, because this is the only amyloidogenic variant, reported until now, with a

FIG. 2. A stereographic overlay of the C-␣ tracing for the Leu 55 3 Pro TTR (brown thick lines) and wild-type TTR (blue thin lines).
The two structures were aligned using the option LSQ of the graphics software package O. Residues 54 -56 (strand D in wild-type TTR) belong to a long exposed loop in Leu 55 3 Pro TTR. The packing interaction between the CE loops (residues 48 -66) of neighboring monomers, belonging to different tetramers, forms the main crystal contact as illustrated in Fig. 3. Additionally, Arg 21 , from the AB loop of one monomer is H-bonded to the ␣-helix residue Gly 83 of the neighboring monomer. Because of this interaction, the side chain of Arg 21 is situated in good electron density, as shown in Fig. 3D. In contrast, Arg 21 is disordered in wild-type TTR, because of inefficient hydrogen bonding (13). In wild-type TTR, Arg 21 , from monomer A, forms a salt bridge to Asp 18 (2.94 Å), a van der Waals interaction with Leu 82 (4.77 Å), and a hydrogen bond to a water molecule with a high B-factor. In monomer B, Arg 21 is modeled in a double conformation. The alternate position of the B21 side chain forms hydrogen bonds to Ser B23 (2.64 Å) and to the hydroxyl group of Tyr B78 (2.43 Å).
In addition to loop CE 7 loop CE, loop AB 7 ␣-helix interactions, there are other interactions, namely Ser 100 (loop FG) 7 Arg 103 (loop FG), Ser 100 (loop FG) 7 Asn 124 (C terminus). These interactions are not observed in the wild-type TTR. Therefore, they are referred as abnormal intersubunit contacts, to distinguish them from the intersubunit contacts that are also observed in wild-type TTR, which are designated as native.
The surface areas involved in the monomer-monomer and dimer-dimer interactions were calculated for the Leu 55 3 Pro and wild-type TTR (Table III). Whereas the intersubunit contacts between two monomers in a dimer do not differ between the variant and wild-type structures, the tetramer dimer-dimer interface diminishes in Leu 55 3 Pro TTR. It is also evident that there is a tight monomer-monomer interaction and a much more tenuous dimer-dimer interface. This fact is experimentally confirmed by the observation that TTR dissociates to form 30-kDa dimers in SDS and 15-kDa monomers only after boiling in SDS with reducing agents (25). Furthermore, the interface between dimers of different tetramers (2524 Å 2 ) is higher than the area of contact between the dimers in a tetramer (1559 and 1837 Å 2 , for the variant and wild-type protein, respectively). Table IV summarizes the Leu 55 3 Pro TTR monomer-monomer hydrogen bonds. DISCUSSION The fibrillar structure resulting from the self-association of an abnormal conformation of TTR is thought to be the causative agent in familial amyloidotic polyneuropathy. However, the mechanism that converts normally soluble TTR tetramers into insoluble amyloid fibrils remains largely unknown.
Structural comparative studies of the native fold and abnormal conformations of TTR variants are expected to be very useful in developing therapeutic strategies for intervention in amyloid disease. In particular, the molecular characterization of the most aggressive TTR amyloidogenic variant, Leu 55 3 Pro TTR, may provide an important contribution to the characterization of the amyloidogenic process.
Studies concerning the kinetics of amyloid formation indicate that the Leu 55 3 Pro TTR variant exists in an amyloidogenic conformation at conditions, whereas the wild-type protein remains stable and non-amyloidogenic. It was observed that amyloid formation from the wild-type protein had an initial rate determining step, not diminished in the presence of electron density map calculated around residue Arg 21 , in Leu 55 3 Pro TTR. The electron density for the side chain of Arg 21 is well defined in Leu 55 3 Pro TTR, because of hydrogen bonding (shown in red) between Arg 21 and Gly 83 from a neighboring monomer. In wild-type TTR, Arg 21 is disordered because of inefficient hydrogen bonding.

TABLE II Hydrogen bonds (Å) involved in tetramer formation
The eight monomers in the Leu 55 3 Pro TTR asymmetric unit cell are referred as A, B, C, D, E, F, G, and H   pre-formed fibrils, which could be associated with the conformational change of the protein into an amyloidogenic intermediate. However, the kinetics of amyloid formation from the Leu 55 3 Pro TTR variant showed the absence of this lag time (26). This could suggest that Leu 55 3 Pro TTR is already in an amyloidogenic conformation, which assembles immediately into amyloid fibrils, under the conditions tested. Furthermore, TTR with a triple deletion on strand D forms amyloid fibrils in vitro at neutral conditions (27).
The evidence for an amyloidogenic conformation for Leu 55 3 Pro TTR is further corroborated by the experimental study of the acid denaturation pathway of some TTR variants, namely Thr 119 3 Met, Val 30 3 Met, and Leu 55 3 Pro. It was observed that the amyloidogenicity of the variants was inversely correlated with the stability of the tetramer toward acid denaturation (26).
Thus, it seems likely that the Leu 55 3 Pro TTR variant exists in an amyloidogenic conformation, with an increased tendency to self-association into amyloid fibrils, even at conditions where the wild-type protein remains stable. It is likely that the crystal structure here described for the Leu 55 3 Pro TTR monomer resembles the amyloidogenic intermediate in the biochemical pathway that leads to the amyloid fibril deposition.
The data reported here indicate that the Leu 55 3 Pro mutation changes the transthyretin secondary structure by the disruption of strand D: residues 54 -56 belong to a long loop that connects ␤-strands C and E, according to the wild-type TTR nomenclature. This loop is involved in the crystallographic packing observed in the present crystal structure (Fig. 3, A and  B).
Positional differences are also detected along the monomermonomer and dimer-dimer interfaces. The hydrogen bonds between the AB loop of one dimer to the strand H of the other dimer (native dimer-dimer interface) are clearly longer than those described for the wild-type TTR tetramer, indicating a potentially less stable Leu 55 3 Pro tetramer (Table II). This result was already pointed out for the amyloidogenic Val 122 3 Ile TTR variant (15), and it is in agreement with biochemical studies concerning the tetramer stability of several TTR variants (23,34). The surface areas associated with the native dimer-dimer interface, i.e. loop AB 7 strand H interaction, which are also an indication of the tetramer stability, are 14.2 and 16.5% of the total dimer surface area for Leu 55 3 Pro TTR and wild-type TTR, respectively.
In addition to the native dimer-dimer intermolecular interactions, which are common to Leu 55 3 Pro and wild-type TTR, there are other interactions between neighboring Leu 55 3 Pro TTR dimers. These include the loop CE 7 loop CE, loop AB 7 ␣-helix, loop FG 7 loop FG. The corresponding area is 23% of the Leu 55 3 Pro TTR dimer total surface area (Table III). This indicates that the abnormal dimer-dimer interface is considerably more extensive than the native dimer-dimer interface, and therefore it may provide the driving force for TTR polymerization into amyloid fibrils. Fig. 3A shows the arrangement of the Leu 55 3 Pro TTR molecules in the unit cell, with the asymmetric units, composed of eight monomers, colored differently. The overall force in the assembly of the units is the intermolecular interaction between the CE loops (residues 48 -66) from the different units (Fig. 3,  A and B) and the hydrogen bonding between the AB loop and the ␣-helix of the nearest neighbor (Fig. 3, C and D). The FG loop (residues 97-103), which differs in all amyloidogenic variants and which is the same in wild-type and non-amyloidogenic TTRs, is also involved in the Leu 55 3 Pro TTR monomer interactions. Ser100, from one monomer, forms hydrogen bonds with Arg 103 or Asn 124 from a neighboring monomer (Table IV). These intermolecular interactions between neighboring Leu 55 3 Pro TTR monomers may be important for the self-association of TTR.
The crystal packing presented in Fig. 3A clearly shows several channels running parallel to each other, thus suggesting a tubular structure that might be similar to the TTR amyloid fibril structure. The protein structure, around each channel, which can be considered as an intermediate structure for TTR amyloid fibril, is composed of four monomers per cross-section. The inner and outer diameters of this structure are 30 and 100 Å, respectively, and its wall is constituted by eight monomers. Four monomers are at one level, and the other four are at a level half way below or above a pseudo-unit cell, with dimension a ϭ 84 Å, along the fibril direction (Fig. 4). The hydrogen bonding direction of the ␤-pleated sheets is about 45°to the proposed fibril direction. In the usual cross-␤ structure, reported for amyloid fibrils, the hydrogen bonding direction is parallel to the fibril axis. However, it is possible that when fibrils are assembled, and assuming that the tetrameric structure was disrupted, the movement between units is not so much restricted, and they may rotate relatively to the fibril axis approaching the situation of the cross-␤ structure.
This model agrees with the results from electron micro- graphs image reconstruction, which showed that TTR amyloid fibrils have a cross-section with four protofilaments arranged around a central hollow core (28). We have now presented information about the contacts between the units in the model, which is important because it provides insight into the development of strategies to inhibit the pathogenic process. Additionally, the reported diameter dimension of the amyloid fibril is similar to the diameter of the tubular structure proposed in this work.
Saraiva et al. (10) reported that the region corresponding to the strand D in Leu 55 3 Pro TTR is exposed, according to monoclonal antibodies binding studies. It is interesting to note that in the Leu 55 3 Pro TTR crystal structure, this region forms the outer part of the asymmetric units that assemble into the tubular structure in the unit cell (Fig. 3, A and B).
We believe that the model presented here, which derives from the analysis of the crystal structure of Leu 55 3 Pro TTR variant, is a good approach to the molecular interactions present in TTR amyloid fibrils. The crystallographic packing analysis indicates putative interactions between units in the TTR amyloid (Table IV, Figs. 3 and 4). In particular, residues Arg 21 , Gly 83 , His 56 , Glu 62 , Ser 100 , Arg 103 , and Asn 124 play an important role in Leu 55 3 Pro TTR intersubunit interface and may be involved in amyloid fibril quaternary interactions.
Site-directed mutagenesis studies, together with amyloid fibril formation in vitro of the constructed mutants, are under way to reinforce and pave the way to the development of therapeutic agents that avoid the adoption of an amyloidogenic conformation.