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Probing the Evolution of Hydroxyisourate Hydrolase into Transthyretin through Active-Site Redesign
J. Mol. Biol. (2011) 409, 504–512
doi:10.1016/j.jmb.2011.04.022 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
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Probing the Evolution of Hydroxyisourate Hydrolase into Transthyretin through Active-Site Redesign Laura Cendron 1,2 , Ileana Ramazzina 3 , Riccardo Percudani 3 , Claudia Rasore 3 , Giuseppe Zanotti 1,2 ⁎ and Rodolfo Berni 3 ⁎ 1
Department of Biological Chemistry, University of Padua, Viale Colombo 3, 35121 Padua, Italy Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129 Padua, Italy 3 Department of Biochemistry and Molecular Biology, University of Parma, Viale G. P. Usberti 23/A, 43100 Parma, Italy 2
Received 14 January 2011; received in revised form 8 April 2011; accepted 10 April 2011 Available online 16 April 2011 Edited by G. Schulz Keywords: 5-hydroxyisourate hydrolase; protein evolution; neofunctionalization; thyroid hormones; transthyretin
5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins. © 2011 Elsevier Ltd. All rights reserved.
*Corresponding authors. G. Zanotti, Department of Biological, Chemistry University of Padua, Viale G. Colombo 3, 35131 Padova, Italy. E-mail addresses: [email protected]; [email protected]. Abbreviations used: HIUase, 5-hydroxyisourate hydrolase; HIU, 5-hydroxyisourate; OHCU, 2-oxo-4hydroxy-4-carboxy-5-ureidoimidazoline; TTR, transthyretin; HBP, halogen binding pocket; PTS, peroxisomal targeting signal.
5-Hydroxyisourate hydrolase (HIUase) is a homotetrameric enzyme that catalyzes the hydrolysis of 5-hydroxyisourate (HIU) into 2-oxo-4-hydroxy-4carboxy-5-ureidoimidazoline (OHCU) within the urate degradation pathway, in which urate, produced in purine metabolism, is converted into (S)-allantoin.1 The components of the complete degradation route from urate into (S)-allantoin, involving the enzymatic activities of urate oxidase, HIUase and OCHU decarboxylase, have recently been identified. 1 HIUase is widely distributed in prokaryotic and eukaryotic lineages. The presence of the gene
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Molecular Evolution of Transthyretin
encoding HIUase (uraH) in a variety of organisms has been found to be associated with the presence of the genes encoding urate oxidase and OHCU decarboxylase.1 In plants, HIUase and OHCU decarboxylase are fused in a bifunctional protein with (S)allantoin synthase activity.2,3 These observations are consistent with a general role for uraH genes in the purine catabolic pathway. An exception is found in enterobacteria, for which the primary function of the uraH gene remains to be clarified.4,5 Notably, a major evolutionary modification in humans and anthropoid apes that affected HIUase, as well as urate oxidase and OHCU decarboxylase,1,6 has been pseudogenization, which resulted in the lack of the urate degradation pathway in such organisms. It has been hypothesized that, in humans, the lack of the pathway, which is associated with high levels of urate in blood, may have beneficial effects due to the antioxidant properties of urate. In mice, in which the pathway is present, the impairment of uric acid metabolism has been found to be deleterious. In fact, an inactivating mutation in HIUase leads to hepatomegaly and hepatocellular carcinoma.7 HIUase is a tetramer formed by the assembly of four identical subunits and characterized by a 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 hydrophobic contacts, to form the tetramer (Fig. 1a). HIUase shares an approximately 50% amino acid sequence similarity with transthyretin (TTR), a well-characterized vertebrate carrier protein participating in the extracellular transport of thyroid hormones and the cotransport of retinol by forming a complex in plasma with retinol binding protein. The two protein families possess a highly conserved fold, as revealed by X-ray analyses of both bacterial5,10,11 and vertebrate (zebrafish)8 HIUases. The similarity of the two protein families can be ascribed to a duplication event in the gene encoding HIUase (uraH),1,12 which gave rise to TTR at a very early stage of vertebrate evolution. In fact, the recent identification of TTR genes in two genera of lamprey13 indicates that gene duplication backdates to before the separation of Agnatha, over 530 million years ago (a phylogenetic tree showing the relationships between HIUase and TTR is presented in Fig. 1b). The long channel that transverses both tetramers of HIUase and TTR, which is coincident with one of their 2-fold symmetry axes (Fig. 1a), was affected by major evolutionary changes. In TTR, this channel harbors the binding sites for two thyroid hormone molecules, which are accommodated in the two symmetrical halves of the channel at the dimer–dimer interface.14,15 On the other hand, in
505 HIUase the channel is partially obstructed at its two ends, where the enzyme active sites occupy only a portion of the thyroxine binding cavities of TTR (Fig. 1c). We report here on the structural comparison of the functional sites of TTR and HIUase, which has suggested that a minimalist redesign of the active site of HIUase may prove effective in shaping a functional site large enough to accommodate the thyroxine molecule. We show that two mutations in HIUase were crucial events for the conversion of the enzyme into TTR. Redesign of the active site of HIUase to generate a thyroxine binding site The prediction of a minimalist reshaping of the active site of HIUase, sufficient to generate a thyroid hormone binding site, was based on the comparison of the structures of zebrafish HIUase,8 the only known structure to date of the enzyme from a vertebrate, and of human TTR. Based on the crystal structure of the thyroxine–TTR complex, 14 the funnel-shaped thyroxine binding site is characterized by three subsites, each composed of pairs of symmetric and hydrophobic halogen binding pockets (HBPs): an outer binding subsite (HBP 1 and 1′), an inner binding subsite (HBP 3 and 3′) and an intervening interface (HBP 2 and 2′). The active site of HIUase, located in a position corresponding to the entrance of the TTR cavity, is lined by residues His12, Asp50, Arg52, His103, Pro105, Leu107, Tyr116, Arg117, Gly118 and Ser119 and symmetry mates, which are highly conserved in a variety of organisms16 (Fig. 2a). A structural comparison of the thyroid hormone binding cavity of TTR with the active site of HIUase provides some insights into how the latter evolved to give rise to the functional site of TTR. Even though 9 out of 10 of the HIUase active-site residues are replaced by nonconservative substitutions in TTR (Fig. 2a), a few amino acid replacements may have been critical with regard to the evolution of HIUase into a binding protein able to bind a ligand markedly bulkier than HIU. The replacement that can be predicted to be most effective in causing the enlargement of the active site of HIUase associated with its evolution into TTR is Y116T (for the numbering systems of HIUase and TTR, see Fig. 2a). In fact, for each of the two active sites of HIUase, defined by the dimer–dimer interface, the side chains of two conserved Tyr116 residues from subunits belonging to the two dimers completely block the access to the internal part of the cavity5,8,10,11 (Fig. 1c) and are replaced by the side chains of two conserved Thr residues (Thr119 and HBP 3 and 3′) in TTR (Fig. 2a). Though several other amino acid differences are present in the functional sites of HIUase and TTR, most of them do not appear to be so critical with regard to the binding of thyroxine. In this respect, His103 and Pro105 of
506
Molecular Evolution of Transthyretin
Fig. 1. Structural and phylogenetic comparison of HIUase and TTR. (a) Ribbon representation of the homotetramer of zebrafish HIUase [Protein Data Bank (PDB) ID: 2H1X8]. The four subunits, in different colors, are conventionally labeled as A, B, C and D. The view is along the molecular 2-fold axis that runs through the central channel of the tetrameric protein (right) or perpendicular to it (left). (b) Phylogenetic tree of HIUase (uraH) and TTR proteins. The maximum-likelihood tree has been constructed with the Proml program of the PHYLIP package and rooted with the midpoint method. Metazoan sequences are indicated with the binomial name of species; branches corresponding to non-metazoan HIUases have been collapsed. The scale bar represents mutations per 100 sites. (c) Left panel: ribbon representation of the zebrafish HIUase tetramer (PDB ID: 2H1X) showing the presence in the central channel of Tyr116 residues (blue) occupying substantially the two active sites at the two ends of the channel. A close-up view of the molecular surface of the active site illustrates the occupancy displayed by the bulky side chains of the two Tyr116 residues (blue). The enzymatic conversion of OHCU into HIU is also shown. (c) Right panel: the same area is depicted for human TTR, in which the binding site for thyroxine (red, stick model; PDB ID: 1ICT9) is not obstructed by the presence of Tyr116 residues. The structural formula of thyroxine is also shown.
HIUase are maintained in the case of lamprey TTR,13 revealing that, in primordial vertebrates, the hormone binding cavity of TTR is reminiscent of the HIUase active site. Interestingly, the side chain
of Leu107 is conserved in both HIUase and TTR (Fig. 2a), but in HIUase, it points toward the space occupied by the hormone in TTR, while in TTR, it points in the opposite direction, thereby leaving
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Fig. 2. Amino acid sequence and functional features of HIUase and mutant forms. (a) Sequence alignment of zebrafish (Danio rerio) HIUase and human TTR. The residues participating in the formation of the active site of zebrafish HIUase8 are shaded in yellow; the residues that are identical in all bona fide TTR sequences13,17 are shaded in orange. Secondary structure elements for zebrafish HIUase (PDB ID: 2H1X) and human TTR (PDB ID: 1F4118) are also shown. (b) Fluorometric in vitro binding assays. Typical titrations of human TTR (black) and of wild-type (cyan), Y116T (green) and Y116T-I16A (red) HIUases with thyroxine; fluorescence intensity is plotted as a function of thyroxine concentration. Conditions: 5 μM TTR or HIUase in 0.05 M sodium phosphate and 0.15 M NaCl, pH 7.2, 37 °C. (c) Enzymatic assays. The kinetics of the enzymatic reaction catalyzed by wild-type (cyan), Y116T (green) and Y116T-I16A (red) HIUases were followed by monitoring the decay of HIU by CDFF spectroscopy at 312 nm. The spontaneous decay of HIU (gray) is also shown. Recombinant wild-type human TTR19 and zebrafish HIUase8 were obtained as described previously. Y116T and Y116T-I16A HIUases were prepared by PCR by using the plasmid pET28b-HIUase as template, a high-fidelity thermostable DNA polymerase (PfuUltra II Fusion HS DNA polymerase; Stratagene) and mutagenic primers complementary to opposite strands. The products of reactions were treated with DpnI (New England Biolabs) to digest the parental DNA template. This procedure allowed us to select the newly synthesized and potentially mutated plasmids. Single clones were then sequenced to confirm the occurrence of the desired mutations. Y116T and Y116T-I16A HIUases were expressed and purified as described for wild-type HIUase.8 An extinction coefficient at 280 nm of 19,940 M− 1 cm− 1 was calculated for Y116T and Y116T-I16A HIUases on the basis of their amino acid sequences. The interaction of L-thyroxine (T4; Sigma-Aldrich) with zebrafish wild-type and Y116T and Y116T-I16A HIUases was investigated by fluorescence titrations by means of a PerkinElmer LS-50B spectrofluorometer, as reported for the titration of human TTR with the hormone,20 monitoring the quenching of the intrinsic protein fluorescence (excitation at 280 nm and emission at 330 nm). Enzymatic activities of zebrafish HIUase and its mutant forms were determined by monitoring the decay of HIU by CD spectroscopy by using a Jasco J715 spectropolarimeter at 25 °C, as described previously;1 CD measurements were carried out at a fixed wavelength (312 nm) in 0.1 M potassium phosphate buffer (final volume of 1 ml in a 10-mm-path-length cuvette), pH 7.6, in the presence of 0.1 mM uric acid, 0.8 U of Candida utilis urate oxidase and 6 μg of recombinant zfHIUase, either wild-type or mutant form. The spontaneous decay of HIU was recorded in the presence of 0.1 mM uric acid and 0.8 U of C. utilis urate oxidase.
space in the cavity to accommodate the hormone. The reason for this critically different orientation is that, in zebrafish HIUase, the rather bulky residue Ile16 blocks, due to its steric hindrance, the position of the side chain of Leu107,8 while in TTR, the
replacing smaller and conserved Ala19 does not have such an effect. Despite the fact that Ile16 is replaced by other residues in the structures of HIUases from bacteria (Gln in the case of Escherichia coli10 and Salmonella dublin5 and Leu in the case of
508 Bacillus subtilis11), these residues occupy the same position and are involved in a similar steric hindrance effect at the enzyme active sites. Moreover, it is worth noting that Ala19 is highly conserved in TTR, consistent with the very important structural and functional role we propose for this residue. An exception is represented in lamprey TTR by Ser19,13 which is anyhow a rather small residue. Based on the analysis above, a single mutant form (Y116T) and a double mutant form (Y116T-I16A) were produced and functionally and structurally characterized to experimentally probe whether two mutations in HIUase could represent crucial and minimalist evolutionary changes sufficient to promote the interaction with thyroxine.
Molecular Evolution of Transthyretin
The kinetics of the enzymatic activities of wildtype HIUase and Y116T and Y116T-I16A HIUases reveal a strong inactivation caused by mutations (Fig. 2c). The rate of hydrolysis of HIU to OHCU catalyzed by HIUase is already drastically reduced (about 99% inactivation) by the single Y116T mutation. This result is not unexpected, as Y116 is the first residue of the ubiquitous C-terminal tetrapeptide YRGS, which represents the signature of HIUases and for which an essential role can therefore be postulated. 16 The double mutant Y116T-I16A HIUase is fully inactive, as the time course of the enzymatic reaction nearly coincides with that of the nonenzymatic decay of HIU, a rather unstable compound susceptible to appreciable hydrolysis in the absence of HIUase.23
Functional divergence of TTR from HIUase To establish whether the Y116T and Y116T-I16A mutations could be critical in the functional evolution of TTR from HIUase, we have assessed their role in the conversion of the enzyme into a binding protein and their influence on the enzymatic activity of HIUase. Binding assays were performed to analyze the interaction of thyroxine with Y116T and Y116T-I16A HIUases. As shown in Fig. 2b, the addition of thyroxine at micromolar concentrations causes a significant quenching of the intrinsic protein fluorescence of Y116T-I16A HIUase, producing a binding curve quite similar to that obtained when human TTR, at equimolar concentration with respect to mutated HIUase, is titrated with the hormone. Although biphasic binding curves, which can be attributed to negative cooperativity in the binding of thyroxine,21 and the lack of well-defined titration end points for both TTR and Y116T-I16A HIUase (Fig. 2b) prevent an accurate analysis of binding parameters, the very similar extent of quenching of protein fluorescence at identical ligand and protein concentrations indicates that the Y116TI16A double mutation generates in HIUase a thyroxine binding site. The mechanism of quenching is likely to be the same, mainly involving the two Trp residues present in HIUase and TTR; moreover, four out of a total of six Tyr residues present in HIUase are also conserved in TTR (Fig. 2a). All these residues, which are located at the same positions in the three-dimensional structures of the two proteins, are similarly distant from the functional sites of HIUase and TTR because of their highly similar fold. Such distances (within 30 Å) are compatible with an energy transfer22 from excited Tyr and Trp residues to the thyroxine molecule, which absorbs (λmax at ∼ 325 nm) in the emission regions of Tyr and Trp residues. A considerably smaller quenching is found, on the other hand, for the single Y116T HIUase mutation, suggesting that the binding cavity is poorly accessible, while a nearly negligible binding is revealed for wild-type HIUase.
Structural basis of the evolutionary divergence of TTR from HIUase The structures of Y116 and Y116T-I16A HIUases show that the introduced mutations do not perturb significantly the protein fold. In fact, the Cα chain traces for the mutated proteins superimpose well on those of wild-type HIUase (Fig. 3a and b legends). Only minor differences can in fact be observed for some flexible loops, that is, residues 34–37 and 59–61. A specific effect of the single Y116T mutation is that of opening the access to the internal part of the thyroxine binding cavity, which is occupied by the bulky side chain of Tyr116 in wild-type HIUase (Fig. 3a). Nevertheless, the cavity where the ligand could be hosted still remains partially occupied by the side chain of Leu107, which corresponds to Leu110 in TTR (Leu110, HBP 2 and 2′ and HBP 3 and 3′). Remarkably, as a result of the substitution at position 16 of the bulky Ile by an Ala residue, in Y116T-I16A HIUase, the side chain of Leu107 is rotated around its Cα–Cβ bond in a way similar to that present in human TTR, thus leaving space for the binding in the central cavity of a bulky ligand, as predicted (Fig. 3b and c). The structure of the thyroxine–Y116T-I16A HIUase complex, obtained from crystals of the mutated enzyme soaked with the hormone, does not show a clear density for the ligand (Fig. 3d), but four maxima for each of the two binding sites of the tetramer can be seen inside the cavity, as also observed in the case of human TTR co-crystallized with the hormone:14 two internal, in a Fourier difference map contoured at the 10-σ level, and two external, in a Fourier difference map contoured at the 5-σ level, respectively. The four peaks we observe are too high, in particular those in the deepest position, to be accounted for by solvent molecules. In addition, an anomalous Fourier difference map calculated with phases from the refined model shows the same peaks, albeit of lower intensity, owing to the fact that data were measured at a wavelength quite far from the absorption edge of
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Fig. 3. Structural details of the reshaping of HIUase active site. (a) Stereo view of a detail of the central channel of the HIUase tetramer (PDB ID: 2H1X) with side chains of residues relevant for mutagenesis explicitly shown. Y116T HIUase (cyan) is superimposed on wild-type HIUase (green; the overall r.m.s.d. for corresponding Cα atoms is 0.451 Å). (b) Same as in (a), with Y116T-I16A HIUase (orange) superimposed on wild-type HIUase (green; the overall r.m.s.d. for corresponding Cα atoms is 0.234 Å). (c) Same as in (b), with Y116T-I16A HIUase (orange) superimposed on human TTR (blue, PDB ID: 1ICT). (d) Stereo view of a detail of the hormone binding cavity in the thyroxine–Y116T-I16A HIUase complex (yellow), with superimposition of the thyroxine molecule as bound to human TTR (PDB ID: 1ICT) (green; only one of the two symmetry-related conformations of the bound hormone is shown). Atoms that we could fit in the electron density map of our structure (four iodine atoms) are shown as small spheres in red. The map was calculated with coefficients (|Fobs| − |Fcalc|), and phases were calculated without ligand and solvent molecules (contour level, 4 σ). The oxygen atom of the hydroxyl group of the hormone, which is visible at a lower contour level, is also shown as a small red sphere. (e) Stereo view of a portion of the anomalous difference electron density map around the hormone binding cavity calculated with phases from the model without the ligand (contour level, 3.5 σ). Data collection and statistics from crystallographic analyses are reported in Table 1.
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Fig. 3 (legend on previous page)
iodine (Fig. 3e). The positions of these peaks fit quite well with those occupied by the four iodine atoms of the TTR-bound thyroxine molecule,9 indicating the binding of the hormone inside the newly generated cavity in the mutated HIUase, despite a possible low occupancy and more than one orientation that might affect the bound ligand. Concluding remarks A wealth of biological data has revealed that adaptation of old proteins for new functions is a common phenomenon in protein evolution, and several examples of homologous enzymes and nonenzymes are known.24 However, it remains to be clarified whether, in the case of drastic changes involving a large proportion of mutated residues, as for the evolution of the HIUase active site into the TTR thyroxine binding site (involving the replacement of 90% of the amino acid residues), a few amino acid replacements may actually be sufficient to trigger protein neofunctionalization. In the case of TTR, neofunctionalization occurred essentially at two levels to shape its role during vertebrate
development. The first level concerns the evolutionary change of the HIUase peroxisomal targeting signal (PTS) into a signal peptide in TTR. In this respect, it should be pointed out that HIUase in mouse and, presumably, in other vertebrates is confined to hepatocytes,7 where the urate degradation pathway takes place. Given the same intron/ exon structures of the genes encoding HIUase and TTR, the first coding exon encodes a signal peptide in TTR and a type 2 PTS in HIUase.12 Notably, conservation of the intron position and sequence similarity in the first exon suggest that, after gene duplication, the PTS sequence of HIUase was transformed into the TTR signal peptide by point mutations.12 These observations, along with the fact that TTR itself is mainly produced by the liver from which it is secreted into the circulation, prompt us to postulate that the TTR ancestor evolved into the plasma carrier TTR through amino acid replacements affecting the first exon of the encoding gene to give rise to a signal peptide, while maintaining the same expression localization. The second level regards the functional divergence of TTR from HIUase. The evolution of HIUase
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Table 1. Statistics on data collection and refinement X-ray data λ (Å), oscillation range (°) Space group, Za Cell parameters a, b, c (Å) β (°) Resolution (Å) Independent reflections Multiplicity Completeness (%) 〈I/σ(I)〉 Rmerge Refinement Total number of atoms, including solvent Mean B value (Å2) Rcryst (%) Rfree (%) Ramachandran plot (%) Most favored Additionally allowed Generously allowed Overall G-factor r.m.s.d. on bond length (Å) and angle (°)
Y116T HIUase
Y116T-I16A HIUase
Y116T-I16A HIUase in complex with thyroxine
0.979, 1 P21, 1
0.983, 0.5 P212121, 2
0.873, 1 P21, 1
45.92, 103.69, 52.63 108.8 44.90–1.70 (1.79–1.70)b 42,791 (5727) 3.5 (3.5) 84.1 (77.9) 10.5 (2.0) 0.051 (0.472)
64.58, 66.59, 236.76 66.52–2.30 (2.32–2.30) 46,362 (6632) 6.9 (7.2) 99.9 (100) 9.3 (6.0) 0.158 (0.446)
45.77, 103.46, 53.49 109.5 51.73–1.95 (2.06–1.95) 33,889 (4721) 2.5 (2.3) 99.0 (94.3) 9.5 (2.1) 0.054 (0.458)
3891 20.0 20.2 (39.2) 25.6 (43.5)
7782 21.1 18.0 (17.9) 24.2 (27.5)
3644 23.6 22.5 (31.7) 27.1 (37.3)
87.3 12.7 0 − 0.2 0.023, 2.0
87.9 12.1 0 − 0.1 0.021, 1.8
87.9 12.1 0 − 0.2 0.021, 1.9
Single crystals of Y116T and Y116T-I16A HIUases were obtained as described for wild-type HIUase.8 Y116T-I16A HIUase in complex with L-thyroxine was obtained by soaking crystals of Y116T-I16A with 0.4 mM hormone. All crystals were analyzed at 100°K without the use of a cryoprotectant solution. The data sets were processed and scaled with MOSFLM and SCALA programs,26 respectively. The crystals of Y116T HIUase and of Y116T-I16A HIUase in complex with thyroxine are isomorphous; they belong to the monoclinic space group P21 and contain one tetramer in the asymmetric unit. The crystals of Y116T-I16A HIUase are orthorhombic, space group P212121, with two tetramers in the asymmetric unit. The structures were solved by molecular replacement with the MOLREP software.27 The refinement was performed using the Refmac program,28 imposing non-crystallographic symmetry restraints on all chains, excluding a few portions where some differences could be observed (residues 34–43, 55–61 and 116–119). The TLS procedure29 was introduced in the last cycles of refinement. The electron density for the mutated residues was clearly visible from the first stages of the refinement. Several cycles of automatic refinement and manual model building reduced the crystallographic R-factor to the final values. The quality of all models, assessed using the PROCHECK software,30 is as expected for structures at this resolution. Data sets for Y116T and Y116T-I16A HIUases were collected at the ID14-4 beamline, and those for the Y116T-I16A HIUase–thyroxine complex were collected at the ID23-2 beamline, European Synchrotron Radiation Facility. a Z indicates the number of HIUase tetramers in the asymmetric unit. b Numbers in parentheses refer to the last resolution shell.
into TTR was accompanied by a number of mutations that have left the overall scaffold unaltered while drastically modifying the protein function. Based on the evidence presented here, we can hypothesize that very few amino acid replacements were originally needed to transform HIUase into a binding protein able to bind rather bulky ligands. After this initial event, which opened the way to the internal cavity of the tetramer and was required to achieve ligand binding ability, abrogating at the same time the catalytic activity of the enzyme, a number of additional mutations were needed to optimize ligand binding and to stabilize the mutated protein. Through the latter mutations, the thyroid hormone binding could thus be definitely established, a situation that is already present in primordial vertebrates, such as lamprey. According to our data based on the rational redesign of the active site of an enzyme, a protein scaffold may be tuned to adapt to a drastically different function by simple walks mainly involving a small number of critical mutations, an evidence that was also
obtained on the basis of directed evolution studies,25 while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins. Accession codes Coordinates and structure factors have been deposited in the Protein Data Bank, as follows: 3IWV for Y116T HIUase, 3IWU for Y116T-I16A HIUase and 3Q1E for Y116T-I16A HIUase in complex with thyroxine.
Acknowledgements We thank the staffs of beamlines ID14-4 and ID232 of European Synchrotron Radiation Facility (Grenoble, France) for technical assistance during data collection. We are also grateful to Roberto Favilla for valuable discussions. This work was supported by the Universities of Parma and Padua, Italy.
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References 1. Ramazzina, I., Folli, C., Secchi, A., Berni, R. & Percudani, R. (2006). Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat. Chem. Biol. 2, 144–148. 2. Lamberto, I., Percudani, R., Gatti, R., Folli, C. & Petrucco, S. (2010). Conserved alternative splicing of Arabidopsis transthyretin-like determines protein localization and S-allantoin synthesis in peroxisomes. Plant Cell, 22, 1564–1574. 3. Pessoa, J., Sárkány, Z., Ferreira-da-Silva, F., Martins, S., Almeida, M. R., Li, J. & Damas, A. M. (2010). Functional characterization of Arabidopsis thaliana transthyretin-like protein. BMC Plant Biol. 10, 30. 4. Hennebry, S. C., Wright, H. M., Likic, V. A. & Richardson, S. J. (2006). Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins, 64, 1024–1045. 5. Hennebry, S. C., Law, R. H., Richardson, S. J., Buckle, A. M. & Whisstock, J. C. (2006). The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J. Mol. Biol. 359, 1389–1399. 6. Keebaugh, A. C. & Thomas, J. W. (2010). The evolutionary fate of the genes encoding the purine catabolic enzymes in hominoids, birds, and reptiles. Mol. Biol. Evol. 27, 1359–1369. 7. Stevenson, W. S., Hyland, C. D., Zhang, J. G., Morgan, P. O., Willson, T. A., Gill, A. et al. (2010). Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice. Proc. Natl Acad. Sci. USA, 107, 16625–16630. 8. Zanotti, G., Cendron, L., Ramazzina, I., Folli, C., Percudani, R. & Berni, R. (2006). Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J. Mol. Biol. 363, 1–9. 9. Wojtczak, A., Neumann, P. & Cody, V. (2001). Structure of a new polymorphic monoclinic form of human transthyretin at 3 Å resolution reveals a mixed complex between unliganded and T4-bound tetramers of TTR. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 957–967. 10. Lundberg, E., Backstrom, S., Sauer, U. H. & SauerEriksson, A. E. (2006). The transthyretin-related protein: structural investigation of a novel protein family. J. Struct. Biol. 155, 445–457. 11. Jung, D. K., Lee, Y., Park, S. G., Park, B. C., Kim, G. H. & Rhee, S. (2006). Structural and functional analysis of PucM, a hydrolase in the ureide pathway and a member of the transthyretin-related protein family. Proc. Natl Acad. Sci. USA, 103, 9790–9795. 12. Zanotti, G., Ramazzina, I., Cendron, L., Folli, C., Percudani, R. & Berni, R. (2009). Vertebrate 5-hydroxyisourate hydrolase identification, function, structure, and evolutionary relationship with transthyretin. In Recent Advances in Transthyretin Evolution, Structure and Biological Functions (Richardson, S. J. & Cody, V., eds), pp. 95–108, Springer, Berlin, Germany. 13. Manzon, R. G., Neuls, T. M. & Manzon, L. A. (2007). Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen. Comp. Endocrinol. 151, 55–65. 14. Wojtczak, A., Cody, V., Luft, J. R. & Pangborn, W. (1996). Structures of human transthyretin complexed
15.
16. 17.
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20. 21.
22. 23. 24. 25. 26.
27. 28.
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30.
with thyroxine at 2.0 Å resolution and 3′,5′-dinitro-Nacetyl-L-thyronine at 2.2 Å resolution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 52, 758–765. Wojtczak, A., Cody, V., Luft, J. R. & Pangborn, W. (2001). Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 Å resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 1061–1070. Eneqvist, T., Lundberg, E., Nilsson, L., Abagyan, R. & Sauer-Eriksson, A. E. (2003). The transthyretin-related protein family. Eur. J. Biochem. 270, 518–532. Zanotti, G., Folli, C., Cendron, L., Alfieri, B., Nishida, S. K., Gliubich, F. et al. (2008). Structural and mutational analyses of protein–protein interactions between transthyretin and retinol-binding protein. FEBS J. 275, 5841–5854. Hörnberg, A., Eneqvist, T., Olofsson, A., Lundgren, E. & Sauer-Eriksson, A. E. (2000). A comparative analysis of 23 structures of the amyloidogenic protein transthyretin. J. Mol. Biol. 302, 649–669. Pasquato, N., Berni, R., Folli, C., Alfieri, B., Cendron, L. & Zanotti, G. (2007). Acidic pH-induced conformational changes in amyloidogenic mutant transthyretin. J. Mol. Biol. 366, 711–719. Nilsson, S. F. & Peterson, P. A. (1971). Evidence for multiple thyroxine-binding sites in human prealbumin. J. Biol. Chem. 246, 6098–6105. Ferguson, R. N., Edelhoch, H., Saroff, H. A., Robbins, J. & Cahnmann, H. J. (1975). Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled 8-anilino-1-naphthalenesulfonic acid. Biochemistry, 14, 282–289. Forster, T. (1967). Mechanism of energy transfer. In Comprehensive Biochemistry (Florkin, M. & Statz, E. H., eds), pp. 61–77, Elsevier, New York, NY. Kahn, K. & Tipton, P. A. (1998). Spectroscopic characterization of intermediates in the urate oxidase reaction. Biochemistry, 37, 11651–11659. Todd, A. E., Orengo, C. A. & Thornton, J. M. (2002). Sequence and structural differences between enzyme and nonenzyme homologs. Structure, 10, 1435–1451. Romero, P. A. & Arnold, F. H. (2009). Exploring protein fitness landscapes by directed evolution. Nat. Rev., Mol. Cell Biol. 10, 866–876. Collaborative Computational Project, Number 4 (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760–763. Vagin, A. A. & Teplyakov, A. (1997). MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1026–1035. Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. (1999). Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr., Sect. D: Biol. Crystallogr. 55, 247–255. Painter, J. & Merritt, E. A. (2006). Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 439–450. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291.
doi:10.1016/j.jmb.2011.04.022 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
COMMUNICATION
Probing the Evolution of Hydroxyisourate Hydrolase into Transthyretin through Active-Site Redesign Laura Cendron 1,2 , Ileana Ramazzina 3 , Riccardo Percudani 3 , Claudia Rasore 3 , Giuseppe Zanotti 1,2 ⁎ and Rodolfo Berni 3 ⁎ 1
Department of Biological Chemistry, University of Padua, Viale Colombo 3, 35121 Padua, Italy Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129 Padua, Italy 3 Department of Biochemistry and Molecular Biology, University of Parma, Viale G. P. Usberti 23/A, 43100 Parma, Italy 2
Received 14 January 2011; received in revised form 8 April 2011; accepted 10 April 2011 Available online 16 April 2011 Edited by G. Schulz Keywords: 5-hydroxyisourate hydrolase; protein evolution; neofunctionalization; thyroid hormones; transthyretin
5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins. © 2011 Elsevier Ltd. All rights reserved.
*Corresponding authors. G. Zanotti, Department of Biological, Chemistry University of Padua, Viale G. Colombo 3, 35131 Padova, Italy. E-mail addresses: [email protected]; [email protected]. Abbreviations used: HIUase, 5-hydroxyisourate hydrolase; HIU, 5-hydroxyisourate; OHCU, 2-oxo-4hydroxy-4-carboxy-5-ureidoimidazoline; TTR, transthyretin; HBP, halogen binding pocket; PTS, peroxisomal targeting signal.
5-Hydroxyisourate hydrolase (HIUase) is a homotetrameric enzyme that catalyzes the hydrolysis of 5-hydroxyisourate (HIU) into 2-oxo-4-hydroxy-4carboxy-5-ureidoimidazoline (OHCU) within the urate degradation pathway, in which urate, produced in purine metabolism, is converted into (S)-allantoin.1 The components of the complete degradation route from urate into (S)-allantoin, involving the enzymatic activities of urate oxidase, HIUase and OCHU decarboxylase, have recently been identified. 1 HIUase is widely distributed in prokaryotic and eukaryotic lineages. The presence of the gene
0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.
Molecular Evolution of Transthyretin
encoding HIUase (uraH) in a variety of organisms has been found to be associated with the presence of the genes encoding urate oxidase and OHCU decarboxylase.1 In plants, HIUase and OHCU decarboxylase are fused in a bifunctional protein with (S)allantoin synthase activity.2,3 These observations are consistent with a general role for uraH genes in the purine catabolic pathway. An exception is found in enterobacteria, for which the primary function of the uraH gene remains to be clarified.4,5 Notably, a major evolutionary modification in humans and anthropoid apes that affected HIUase, as well as urate oxidase and OHCU decarboxylase,1,6 has been pseudogenization, which resulted in the lack of the urate degradation pathway in such organisms. It has been hypothesized that, in humans, the lack of the pathway, which is associated with high levels of urate in blood, may have beneficial effects due to the antioxidant properties of urate. In mice, in which the pathway is present, the impairment of uric acid metabolism has been found to be deleterious. In fact, an inactivating mutation in HIUase leads to hepatomegaly and hepatocellular carcinoma.7 HIUase is a tetramer formed by the assembly of four identical subunits and characterized by a 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 hydrophobic contacts, to form the tetramer (Fig. 1a). HIUase shares an approximately 50% amino acid sequence similarity with transthyretin (TTR), a well-characterized vertebrate carrier protein participating in the extracellular transport of thyroid hormones and the cotransport of retinol by forming a complex in plasma with retinol binding protein. The two protein families possess a highly conserved fold, as revealed by X-ray analyses of both bacterial5,10,11 and vertebrate (zebrafish)8 HIUases. The similarity of the two protein families can be ascribed to a duplication event in the gene encoding HIUase (uraH),1,12 which gave rise to TTR at a very early stage of vertebrate evolution. In fact, the recent identification of TTR genes in two genera of lamprey13 indicates that gene duplication backdates to before the separation of Agnatha, over 530 million years ago (a phylogenetic tree showing the relationships between HIUase and TTR is presented in Fig. 1b). The long channel that transverses both tetramers of HIUase and TTR, which is coincident with one of their 2-fold symmetry axes (Fig. 1a), was affected by major evolutionary changes. In TTR, this channel harbors the binding sites for two thyroid hormone molecules, which are accommodated in the two symmetrical halves of the channel at the dimer–dimer interface.14,15 On the other hand, in
505 HIUase the channel is partially obstructed at its two ends, where the enzyme active sites occupy only a portion of the thyroxine binding cavities of TTR (Fig. 1c). We report here on the structural comparison of the functional sites of TTR and HIUase, which has suggested that a minimalist redesign of the active site of HIUase may prove effective in shaping a functional site large enough to accommodate the thyroxine molecule. We show that two mutations in HIUase were crucial events for the conversion of the enzyme into TTR. Redesign of the active site of HIUase to generate a thyroxine binding site The prediction of a minimalist reshaping of the active site of HIUase, sufficient to generate a thyroid hormone binding site, was based on the comparison of the structures of zebrafish HIUase,8 the only known structure to date of the enzyme from a vertebrate, and of human TTR. Based on the crystal structure of the thyroxine–TTR complex, 14 the funnel-shaped thyroxine binding site is characterized by three subsites, each composed of pairs of symmetric and hydrophobic halogen binding pockets (HBPs): an outer binding subsite (HBP 1 and 1′), an inner binding subsite (HBP 3 and 3′) and an intervening interface (HBP 2 and 2′). The active site of HIUase, located in a position corresponding to the entrance of the TTR cavity, is lined by residues His12, Asp50, Arg52, His103, Pro105, Leu107, Tyr116, Arg117, Gly118 and Ser119 and symmetry mates, which are highly conserved in a variety of organisms16 (Fig. 2a). A structural comparison of the thyroid hormone binding cavity of TTR with the active site of HIUase provides some insights into how the latter evolved to give rise to the functional site of TTR. Even though 9 out of 10 of the HIUase active-site residues are replaced by nonconservative substitutions in TTR (Fig. 2a), a few amino acid replacements may have been critical with regard to the evolution of HIUase into a binding protein able to bind a ligand markedly bulkier than HIU. The replacement that can be predicted to be most effective in causing the enlargement of the active site of HIUase associated with its evolution into TTR is Y116T (for the numbering systems of HIUase and TTR, see Fig. 2a). In fact, for each of the two active sites of HIUase, defined by the dimer–dimer interface, the side chains of two conserved Tyr116 residues from subunits belonging to the two dimers completely block the access to the internal part of the cavity5,8,10,11 (Fig. 1c) and are replaced by the side chains of two conserved Thr residues (Thr119 and HBP 3 and 3′) in TTR (Fig. 2a). Though several other amino acid differences are present in the functional sites of HIUase and TTR, most of them do not appear to be so critical with regard to the binding of thyroxine. In this respect, His103 and Pro105 of
506
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Fig. 1. Structural and phylogenetic comparison of HIUase and TTR. (a) Ribbon representation of the homotetramer of zebrafish HIUase [Protein Data Bank (PDB) ID: 2H1X8]. The four subunits, in different colors, are conventionally labeled as A, B, C and D. The view is along the molecular 2-fold axis that runs through the central channel of the tetrameric protein (right) or perpendicular to it (left). (b) Phylogenetic tree of HIUase (uraH) and TTR proteins. The maximum-likelihood tree has been constructed with the Proml program of the PHYLIP package and rooted with the midpoint method. Metazoan sequences are indicated with the binomial name of species; branches corresponding to non-metazoan HIUases have been collapsed. The scale bar represents mutations per 100 sites. (c) Left panel: ribbon representation of the zebrafish HIUase tetramer (PDB ID: 2H1X) showing the presence in the central channel of Tyr116 residues (blue) occupying substantially the two active sites at the two ends of the channel. A close-up view of the molecular surface of the active site illustrates the occupancy displayed by the bulky side chains of the two Tyr116 residues (blue). The enzymatic conversion of OHCU into HIU is also shown. (c) Right panel: the same area is depicted for human TTR, in which the binding site for thyroxine (red, stick model; PDB ID: 1ICT9) is not obstructed by the presence of Tyr116 residues. The structural formula of thyroxine is also shown.
HIUase are maintained in the case of lamprey TTR,13 revealing that, in primordial vertebrates, the hormone binding cavity of TTR is reminiscent of the HIUase active site. Interestingly, the side chain
of Leu107 is conserved in both HIUase and TTR (Fig. 2a), but in HIUase, it points toward the space occupied by the hormone in TTR, while in TTR, it points in the opposite direction, thereby leaving
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Fig. 2. Amino acid sequence and functional features of HIUase and mutant forms. (a) Sequence alignment of zebrafish (Danio rerio) HIUase and human TTR. The residues participating in the formation of the active site of zebrafish HIUase8 are shaded in yellow; the residues that are identical in all bona fide TTR sequences13,17 are shaded in orange. Secondary structure elements for zebrafish HIUase (PDB ID: 2H1X) and human TTR (PDB ID: 1F4118) are also shown. (b) Fluorometric in vitro binding assays. Typical titrations of human TTR (black) and of wild-type (cyan), Y116T (green) and Y116T-I16A (red) HIUases with thyroxine; fluorescence intensity is plotted as a function of thyroxine concentration. Conditions: 5 μM TTR or HIUase in 0.05 M sodium phosphate and 0.15 M NaCl, pH 7.2, 37 °C. (c) Enzymatic assays. The kinetics of the enzymatic reaction catalyzed by wild-type (cyan), Y116T (green) and Y116T-I16A (red) HIUases were followed by monitoring the decay of HIU by CDFF spectroscopy at 312 nm. The spontaneous decay of HIU (gray) is also shown. Recombinant wild-type human TTR19 and zebrafish HIUase8 were obtained as described previously. Y116T and Y116T-I16A HIUases were prepared by PCR by using the plasmid pET28b-HIUase as template, a high-fidelity thermostable DNA polymerase (PfuUltra II Fusion HS DNA polymerase; Stratagene) and mutagenic primers complementary to opposite strands. The products of reactions were treated with DpnI (New England Biolabs) to digest the parental DNA template. This procedure allowed us to select the newly synthesized and potentially mutated plasmids. Single clones were then sequenced to confirm the occurrence of the desired mutations. Y116T and Y116T-I16A HIUases were expressed and purified as described for wild-type HIUase.8 An extinction coefficient at 280 nm of 19,940 M− 1 cm− 1 was calculated for Y116T and Y116T-I16A HIUases on the basis of their amino acid sequences. The interaction of L-thyroxine (T4; Sigma-Aldrich) with zebrafish wild-type and Y116T and Y116T-I16A HIUases was investigated by fluorescence titrations by means of a PerkinElmer LS-50B spectrofluorometer, as reported for the titration of human TTR with the hormone,20 monitoring the quenching of the intrinsic protein fluorescence (excitation at 280 nm and emission at 330 nm). Enzymatic activities of zebrafish HIUase and its mutant forms were determined by monitoring the decay of HIU by CD spectroscopy by using a Jasco J715 spectropolarimeter at 25 °C, as described previously;1 CD measurements were carried out at a fixed wavelength (312 nm) in 0.1 M potassium phosphate buffer (final volume of 1 ml in a 10-mm-path-length cuvette), pH 7.6, in the presence of 0.1 mM uric acid, 0.8 U of Candida utilis urate oxidase and 6 μg of recombinant zfHIUase, either wild-type or mutant form. The spontaneous decay of HIU was recorded in the presence of 0.1 mM uric acid and 0.8 U of C. utilis urate oxidase.
space in the cavity to accommodate the hormone. The reason for this critically different orientation is that, in zebrafish HIUase, the rather bulky residue Ile16 blocks, due to its steric hindrance, the position of the side chain of Leu107,8 while in TTR, the
replacing smaller and conserved Ala19 does not have such an effect. Despite the fact that Ile16 is replaced by other residues in the structures of HIUases from bacteria (Gln in the case of Escherichia coli10 and Salmonella dublin5 and Leu in the case of
508 Bacillus subtilis11), these residues occupy the same position and are involved in a similar steric hindrance effect at the enzyme active sites. Moreover, it is worth noting that Ala19 is highly conserved in TTR, consistent with the very important structural and functional role we propose for this residue. An exception is represented in lamprey TTR by Ser19,13 which is anyhow a rather small residue. Based on the analysis above, a single mutant form (Y116T) and a double mutant form (Y116T-I16A) were produced and functionally and structurally characterized to experimentally probe whether two mutations in HIUase could represent crucial and minimalist evolutionary changes sufficient to promote the interaction with thyroxine.
Molecular Evolution of Transthyretin
The kinetics of the enzymatic activities of wildtype HIUase and Y116T and Y116T-I16A HIUases reveal a strong inactivation caused by mutations (Fig. 2c). The rate of hydrolysis of HIU to OHCU catalyzed by HIUase is already drastically reduced (about 99% inactivation) by the single Y116T mutation. This result is not unexpected, as Y116 is the first residue of the ubiquitous C-terminal tetrapeptide YRGS, which represents the signature of HIUases and for which an essential role can therefore be postulated. 16 The double mutant Y116T-I16A HIUase is fully inactive, as the time course of the enzymatic reaction nearly coincides with that of the nonenzymatic decay of HIU, a rather unstable compound susceptible to appreciable hydrolysis in the absence of HIUase.23
Functional divergence of TTR from HIUase To establish whether the Y116T and Y116T-I16A mutations could be critical in the functional evolution of TTR from HIUase, we have assessed their role in the conversion of the enzyme into a binding protein and their influence on the enzymatic activity of HIUase. Binding assays were performed to analyze the interaction of thyroxine with Y116T and Y116T-I16A HIUases. As shown in Fig. 2b, the addition of thyroxine at micromolar concentrations causes a significant quenching of the intrinsic protein fluorescence of Y116T-I16A HIUase, producing a binding curve quite similar to that obtained when human TTR, at equimolar concentration with respect to mutated HIUase, is titrated with the hormone. Although biphasic binding curves, which can be attributed to negative cooperativity in the binding of thyroxine,21 and the lack of well-defined titration end points for both TTR and Y116T-I16A HIUase (Fig. 2b) prevent an accurate analysis of binding parameters, the very similar extent of quenching of protein fluorescence at identical ligand and protein concentrations indicates that the Y116TI16A double mutation generates in HIUase a thyroxine binding site. The mechanism of quenching is likely to be the same, mainly involving the two Trp residues present in HIUase and TTR; moreover, four out of a total of six Tyr residues present in HIUase are also conserved in TTR (Fig. 2a). All these residues, which are located at the same positions in the three-dimensional structures of the two proteins, are similarly distant from the functional sites of HIUase and TTR because of their highly similar fold. Such distances (within 30 Å) are compatible with an energy transfer22 from excited Tyr and Trp residues to the thyroxine molecule, which absorbs (λmax at ∼ 325 nm) in the emission regions of Tyr and Trp residues. A considerably smaller quenching is found, on the other hand, for the single Y116T HIUase mutation, suggesting that the binding cavity is poorly accessible, while a nearly negligible binding is revealed for wild-type HIUase.
Structural basis of the evolutionary divergence of TTR from HIUase The structures of Y116 and Y116T-I16A HIUases show that the introduced mutations do not perturb significantly the protein fold. In fact, the Cα chain traces for the mutated proteins superimpose well on those of wild-type HIUase (Fig. 3a and b legends). Only minor differences can in fact be observed for some flexible loops, that is, residues 34–37 and 59–61. A specific effect of the single Y116T mutation is that of opening the access to the internal part of the thyroxine binding cavity, which is occupied by the bulky side chain of Tyr116 in wild-type HIUase (Fig. 3a). Nevertheless, the cavity where the ligand could be hosted still remains partially occupied by the side chain of Leu107, which corresponds to Leu110 in TTR (Leu110, HBP 2 and 2′ and HBP 3 and 3′). Remarkably, as a result of the substitution at position 16 of the bulky Ile by an Ala residue, in Y116T-I16A HIUase, the side chain of Leu107 is rotated around its Cα–Cβ bond in a way similar to that present in human TTR, thus leaving space for the binding in the central cavity of a bulky ligand, as predicted (Fig. 3b and c). The structure of the thyroxine–Y116T-I16A HIUase complex, obtained from crystals of the mutated enzyme soaked with the hormone, does not show a clear density for the ligand (Fig. 3d), but four maxima for each of the two binding sites of the tetramer can be seen inside the cavity, as also observed in the case of human TTR co-crystallized with the hormone:14 two internal, in a Fourier difference map contoured at the 10-σ level, and two external, in a Fourier difference map contoured at the 5-σ level, respectively. The four peaks we observe are too high, in particular those in the deepest position, to be accounted for by solvent molecules. In addition, an anomalous Fourier difference map calculated with phases from the refined model shows the same peaks, albeit of lower intensity, owing to the fact that data were measured at a wavelength quite far from the absorption edge of
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Fig. 3. Structural details of the reshaping of HIUase active site. (a) Stereo view of a detail of the central channel of the HIUase tetramer (PDB ID: 2H1X) with side chains of residues relevant for mutagenesis explicitly shown. Y116T HIUase (cyan) is superimposed on wild-type HIUase (green; the overall r.m.s.d. for corresponding Cα atoms is 0.451 Å). (b) Same as in (a), with Y116T-I16A HIUase (orange) superimposed on wild-type HIUase (green; the overall r.m.s.d. for corresponding Cα atoms is 0.234 Å). (c) Same as in (b), with Y116T-I16A HIUase (orange) superimposed on human TTR (blue, PDB ID: 1ICT). (d) Stereo view of a detail of the hormone binding cavity in the thyroxine–Y116T-I16A HIUase complex (yellow), with superimposition of the thyroxine molecule as bound to human TTR (PDB ID: 1ICT) (green; only one of the two symmetry-related conformations of the bound hormone is shown). Atoms that we could fit in the electron density map of our structure (four iodine atoms) are shown as small spheres in red. The map was calculated with coefficients (|Fobs| − |Fcalc|), and phases were calculated without ligand and solvent molecules (contour level, 4 σ). The oxygen atom of the hydroxyl group of the hormone, which is visible at a lower contour level, is also shown as a small red sphere. (e) Stereo view of a portion of the anomalous difference electron density map around the hormone binding cavity calculated with phases from the model without the ligand (contour level, 3.5 σ). Data collection and statistics from crystallographic analyses are reported in Table 1.
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Fig. 3 (legend on previous page)
iodine (Fig. 3e). The positions of these peaks fit quite well with those occupied by the four iodine atoms of the TTR-bound thyroxine molecule,9 indicating the binding of the hormone inside the newly generated cavity in the mutated HIUase, despite a possible low occupancy and more than one orientation that might affect the bound ligand. Concluding remarks A wealth of biological data has revealed that adaptation of old proteins for new functions is a common phenomenon in protein evolution, and several examples of homologous enzymes and nonenzymes are known.24 However, it remains to be clarified whether, in the case of drastic changes involving a large proportion of mutated residues, as for the evolution of the HIUase active site into the TTR thyroxine binding site (involving the replacement of 90% of the amino acid residues), a few amino acid replacements may actually be sufficient to trigger protein neofunctionalization. In the case of TTR, neofunctionalization occurred essentially at two levels to shape its role during vertebrate
development. The first level concerns the evolutionary change of the HIUase peroxisomal targeting signal (PTS) into a signal peptide in TTR. In this respect, it should be pointed out that HIUase in mouse and, presumably, in other vertebrates is confined to hepatocytes,7 where the urate degradation pathway takes place. Given the same intron/ exon structures of the genes encoding HIUase and TTR, the first coding exon encodes a signal peptide in TTR and a type 2 PTS in HIUase.12 Notably, conservation of the intron position and sequence similarity in the first exon suggest that, after gene duplication, the PTS sequence of HIUase was transformed into the TTR signal peptide by point mutations.12 These observations, along with the fact that TTR itself is mainly produced by the liver from which it is secreted into the circulation, prompt us to postulate that the TTR ancestor evolved into the plasma carrier TTR through amino acid replacements affecting the first exon of the encoding gene to give rise to a signal peptide, while maintaining the same expression localization. The second level regards the functional divergence of TTR from HIUase. The evolution of HIUase
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Table 1. Statistics on data collection and refinement X-ray data λ (Å), oscillation range (°) Space group, Za Cell parameters a, b, c (Å) β (°) Resolution (Å) Independent reflections Multiplicity Completeness (%) 〈I/σ(I)〉 Rmerge Refinement Total number of atoms, including solvent Mean B value (Å2) Rcryst (%) Rfree (%) Ramachandran plot (%) Most favored Additionally allowed Generously allowed Overall G-factor r.m.s.d. on bond length (Å) and angle (°)
Y116T HIUase
Y116T-I16A HIUase
Y116T-I16A HIUase in complex with thyroxine
0.979, 1 P21, 1
0.983, 0.5 P212121, 2
0.873, 1 P21, 1
45.92, 103.69, 52.63 108.8 44.90–1.70 (1.79–1.70)b 42,791 (5727) 3.5 (3.5) 84.1 (77.9) 10.5 (2.0) 0.051 (0.472)
64.58, 66.59, 236.76 66.52–2.30 (2.32–2.30) 46,362 (6632) 6.9 (7.2) 99.9 (100) 9.3 (6.0) 0.158 (0.446)
45.77, 103.46, 53.49 109.5 51.73–1.95 (2.06–1.95) 33,889 (4721) 2.5 (2.3) 99.0 (94.3) 9.5 (2.1) 0.054 (0.458)
3891 20.0 20.2 (39.2) 25.6 (43.5)
7782 21.1 18.0 (17.9) 24.2 (27.5)
3644 23.6 22.5 (31.7) 27.1 (37.3)
87.3 12.7 0 − 0.2 0.023, 2.0
87.9 12.1 0 − 0.1 0.021, 1.8
87.9 12.1 0 − 0.2 0.021, 1.9
Single crystals of Y116T and Y116T-I16A HIUases were obtained as described for wild-type HIUase.8 Y116T-I16A HIUase in complex with L-thyroxine was obtained by soaking crystals of Y116T-I16A with 0.4 mM hormone. All crystals were analyzed at 100°K without the use of a cryoprotectant solution. The data sets were processed and scaled with MOSFLM and SCALA programs,26 respectively. The crystals of Y116T HIUase and of Y116T-I16A HIUase in complex with thyroxine are isomorphous; they belong to the monoclinic space group P21 and contain one tetramer in the asymmetric unit. The crystals of Y116T-I16A HIUase are orthorhombic, space group P212121, with two tetramers in the asymmetric unit. The structures were solved by molecular replacement with the MOLREP software.27 The refinement was performed using the Refmac program,28 imposing non-crystallographic symmetry restraints on all chains, excluding a few portions where some differences could be observed (residues 34–43, 55–61 and 116–119). The TLS procedure29 was introduced in the last cycles of refinement. The electron density for the mutated residues was clearly visible from the first stages of the refinement. Several cycles of automatic refinement and manual model building reduced the crystallographic R-factor to the final values. The quality of all models, assessed using the PROCHECK software,30 is as expected for structures at this resolution. Data sets for Y116T and Y116T-I16A HIUases were collected at the ID14-4 beamline, and those for the Y116T-I16A HIUase–thyroxine complex were collected at the ID23-2 beamline, European Synchrotron Radiation Facility. a Z indicates the number of HIUase tetramers in the asymmetric unit. b Numbers in parentheses refer to the last resolution shell.
into TTR was accompanied by a number of mutations that have left the overall scaffold unaltered while drastically modifying the protein function. Based on the evidence presented here, we can hypothesize that very few amino acid replacements were originally needed to transform HIUase into a binding protein able to bind rather bulky ligands. After this initial event, which opened the way to the internal cavity of the tetramer and was required to achieve ligand binding ability, abrogating at the same time the catalytic activity of the enzyme, a number of additional mutations were needed to optimize ligand binding and to stabilize the mutated protein. Through the latter mutations, the thyroid hormone binding could thus be definitely established, a situation that is already present in primordial vertebrates, such as lamprey. According to our data based on the rational redesign of the active site of an enzyme, a protein scaffold may be tuned to adapt to a drastically different function by simple walks mainly involving a small number of critical mutations, an evidence that was also
obtained on the basis of directed evolution studies,25 while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins. Accession codes Coordinates and structure factors have been deposited in the Protein Data Bank, as follows: 3IWV for Y116T HIUase, 3IWU for Y116T-I16A HIUase and 3Q1E for Y116T-I16A HIUase in complex with thyroxine.
Acknowledgements We thank the staffs of beamlines ID14-4 and ID232 of European Synchrotron Radiation Facility (Grenoble, France) for technical assistance during data collection. We are also grateful to Roberto Favilla for valuable discussions. This work was supported by the Universities of Parma and Padua, Italy.
Molecular Evolution of Transthyretin
512
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