Báo cáo khoa học: Structural and mutational analyses of protein–protein interactions between transthyretin and retinol-binding protein doc

Structural and mutational analyses of protein–protein interactions between transthyretin and retinol-binding protein Giuseppe Zanotti1,2, Claudia Folli3, Laura Cendron1,2, Beatrice Alfieri3, Sonia K Nishida4, Francesca Gliubich1,*, Nicola Pasquato1, Alessandro Negro5 and Rodolfo Berni3 Department of Chemical Sciences and Institute of Biomolecular Chemistry-CNR, University of Padua, Italy Venetian Institute of Molecular Medicine, Padua, Italy Department of Biochemistry and Molecular Biology, University of Parma, Italy Department of Medicine, Federal University of Sa Paulo, Brazil ˜o Department of Experimental Veterinary Sciences and CRIBI, University of Padua, Italy Keywords Fab; mutational analysis; protein–protein interactions; retinol-binding protein; transthyretin Correspondence G Zanotti, Department of Chemical Sciences, University of Padua, Via Marzolo 1, 35131 Padua, Italy Fax: +39 049 8275239 Tel: +39 049 8275245 E-mail: giuseppe.zanotti@unipd.it R Berni, Department of Biochemistry and Molecular Biology, University of Parma, V.le G.P Usberti 23 ⁄ A, 43100 Parma, Italy Fax: +39 0521 905151 Tel: +39 0521 905645 E-mail: rodolfo.berni@unipr.it *Present address KCL Enterprises Ltd, London, UK Database Atomic coordinates and structure factors have been deposited at the Protein Data Bank (PDB) (http://www.rcsb.org) for immediate release: PDB 3BSZ for the TTR–RBP– Fab complex and PDB 3BT0 and 3CXF for the V20S and Y114H TTR variants, respectively Transthyretin is a tetrameric binding protein involved in the transport of thyroid hormones and in the cotransport of retinol by forming a complex in plasma with retinol-binding protein In the present study, we report the crystal structure of a macromolecular complex, in which human transthyretin, human holo-retinol-binding protein and a murine anti-retinol-binding protein Fab are assembled according to a : : stoichiometry The main interactions, both polar and apolar, between retinol-binding protein and transthyretin involve the retinol hydroxyl group and a limited number of solvent exposed residues The relevance of transthyretin residues in complex formation with retinol-binding protein has been examined by mutational analysis, and the structural consequences of some transthyretin point mutations affecting protein–protein recognition have been investigated Despite a few exceptions, in general, the substitution of a hydrophilic for a hydrophobic side chain in contact regions results in a decrease or even a loss of binding affinity, thus revealing the importance of interfacial hydrophobic interactions and a high degree of complementarity between retinolbinding protein and transthyretin The effect is particularly evident when the mutation affects an interacting residue present in two distinct subunits of transthyretin participating simultaneously in two interactions with a retinol-binding protein molecule This is the case of the amyloidogenic I84S replacement, which abolishes the interaction with retinol-binding protein and is associated with an altered retinol-binding protein plasma transport in carriers of this mutation Remarkably, some of the residues in mutated human transthyretin that weaken or abolish the interaction with retinolbinding protein are present in piscine transthyretin, consistent with the lack of interaction between retinol-binding protein and transthyretin in fish (Received 23 July 2008, revised 22 September 2008, accepted 25 September 2008) doi:10.1111/j.1742-4658.2008.06705.x Abbreviations PDB, Protein Data Bank; RBP, retinol-binding protein; TTR, transthyretin FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS 5841 Transthyretin–retinol-binding protein interactions G Zanotti et al Transthyretin (TTR), a homotetramer of approximately 55 kDa, is a thyroid hormone-binding protein present in the extracellular fluids of vertebrates, where it participates, together with other binding proteins, in the distribution of thyroid hormones (thyroxine and triiodothyronine) [1] It was generated during early vertebrate evolution as a result of a duplication event in the gene encoding 5-hydroxyisourate hydrolase, an enzyme distributed in several prokaryotes and in several eukaryotic lineages and involved in purine metabolism [2–6] The extracellular transport of retinol (vitamin A alcohol) is specifically mediated by retinol-binding protein (RBP, also known as RBP 4), a monomeric protein of 21 kDa that delivers the vitamin molecule to the target cells [7,8], where a membrane RBP receptor represents a major mediator of cellular vitamin A uptake [9,10] TTR and RBP are synthesized primarily by the hepatocytes and are secreted into the circulation, where RBP is found bound to TTR The association of RBP with TTR increases the stability of the retinol–RBP complex [11,12] and, according to various lines of evidence [13–15], is believed to reduce the glomerular filtration of the relatively small RBP molecule In turn, the stability of the RBP–TTR complex is strongly affected by the presence of retinol bound to RBP within the complex, a feature that is believed to be of physiological significance The affinity of holoRBP for TTR is significantly higher than that of apoRBP [16,17], which is consistent with holoRBP being retained in the circulation as the protein–protein complex and with the uncomplexed apoRBP molecule, resulting from the delivery of retinol, being selectively cleared from the circulation by glomerular filtration TTR has been associated with human diseases It is one of several proteins that can produce the extracellular accumulation in tissues of protein aggregates, in the form of fibrils, which are responsible for degenerative diseases known as amyloidoses; to date, more than 100 point mutations have been described for TTR, most of which are involved in familial amyloidoses [18,19] Moreover, a protective role of TTR in Alzheimer’s disease has recently been proposed [20,21] RBP is an adipocyte-derived ‘signal’ that may contribute to the pathogenesis of insulin resistance [22] Well-refined crystal structures of TTR from different vertebrate species, including mammals [23–25], chicken [26] and fish (sea bream) [27,28], and of RBP from mammals [29–33] and chicken [34], have been described The crystal structures of heterologous (human TTR–chicken RBP) [35] and homologous (human TTR–human RBP) [36] TTR–holoRBP com5842 plexes have also been determined, both characterized by a : TTR : RBP stoichiometry in which each TTR-bound RBP molecule interacts simultaneously with three TTR subunits [35,36] TTR is a tetrameric protein formed by the assembly of four identical subunits Each monomer is composed of eight anti-parallel b-strands (A–H), arranged in a topology similar to the Greek key b-barrel, with a short a-helix located at the end of b-strand E In the tetramer, the four monomers are organized as a dimer of dimers Specifically, two monomers are held together to form a stable dimer through a net of H-bond interactions involving the two edge b-strands H and F To form the tetramer, two dimers associate back to back, mainly through hydrophobic contacts between residues of the loops formed by b-strands A and B and b-strands G and H One of the two-fold symmetry axes of the tetramer is coincident with a long channel that transverses the entire molecule and harbors two binding sites for thyroid hormones RBP is a single domain protein, made up of an N-terminal coil, eight anti-parallel b-strands (A–H) and a short a-helix close to the C-terminus The core of the protein is the internal cavity of an eightstranded up-and-down b-barrel The vitamin molecule is accommodated within the cavity of the barrel: the b-ionone ring is innermost, the polyene chain is fully extended and the hydroxyl end group is almost solvent exposed, in the region of the loops that connect b-strands A and B, C and D and E and F and surround the entrance of the b-barrel at the open end of the cavity As a result of evolutionary restraints imposed by the multiple interactions established by both RBP and TTR, a high degree of structural similarity appears to be preserved for these two proteins from phylogenetically distant vertebrate species It should be noted, however, that piscine TTR and RBP lack the ability to form a protein– protein complex [27] The molecular basis of the evolution of the two piscine proteins into proteins that possess the ability to interact with each other in terrestrial vertebrates remains to be clarified In the present study, we report on the structure of a complex formed by the association of human TTR, human holoRBP and a murine anti-RBP Fab, and on a mutational analysis of the RBP-binding determinants present in the human TTR molecule The data provide insight into the molecular basis of the altered plasma transport of RBP in carriers of a relevant amyloidogenic TTR mutation (I84S) and of the changes in the TTR molecule that have affected its ability to interact with RBP during the course of vertebrate evolution FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS G Zanotti et al Results Structure of a complex between human TTR, human holoRBP and an anti-RBP Fab The crystal structure of the human TTR–RBP complex bound to an anti-RBP Fab could be determined ˚ at a resolution of 3.36 A, similar to that obtained for the crystal structures of TTR–RBP complexes [35,36] The molecular model, obtained by molecular replacement starting from the available high resolution crystal structures of RBP and TTR, is of reasonable quality The anti-RBP Fab is bound to an RBP epitope that is well separated from the TTR-binding determinants present in the RBP molecule It interacts with RBP on the side opposite to that involved in the binding to TTR (Fig 1A), so that the interactions between RBP and TTR are not affected by the RBP-bound Fab The entire complex, composed of one TTR tetramer and two holoRBP molecules in complex with Fab, is present in the asymmetric unit (Fig 1A) The two RBP–Fab sub-complexes are arranged symmetrically around the two-fold axis of TTR running through the central channel that transverses the TTR molecule RBP and TTR substantially maintain the structure they have in the uncomplexed state, with only minor changes in contact regions The region of entrance of retinol into the b-barrel cavity of RBP (i.e loops 32–36, 63–67 and 92–98), and retinol itself, participate in the recognition of TTR, involving residues that are essentially located in loops connecting b-strands of the tetrameric protein, in such a way that one RBP molecule interacts simultaneously with three TTR subunits (Table and Fig 1A,B) The RBP–TTR interactions are both polar or apolar (Table 1) The contact surface of TTR is characterized by a prevalence of hydrophobic residues in the case of the subunits B and C and of hydrophilic residues in the case of the subunit A Val20, Trp79, Leu82, Ile84, Pro113 and Tyr114 from both subunits B and C of TTR form a hydrophobic patch, which is in contact in the protein–protein complex with the hydrophobic patch formed by the residues Trp67, Leu63, Leu64, Val69, Phe96 and Leu97 of RBP Instead, the interacting hydrophilic residues from the subunit A of TTR are closer to the border of the contact surface of TTR Four H-bonds, mainly between RBP and the subunit B of TTR, are also present (Table 1) The area buried on the two pro˚ teins upon complex formation is 1443 A2, out of a ˚ total area of 9307 and 21502 A2 for the two separated RBP and TTR molecules, respectively Transthyretin–retinol-binding protein interactions The Fab–RBP interaction involves the region preceding the a-helix and the C-terminal b-strand of RBP and the hyper-variable regions of Fab: loops 53–56 and 100–103 and the short helix 28–32 of chain H and loops 31–36 and 53–56 of chain L The interactions, which are mainly polar and comprise several H-bonds, are summarized in Table The number of residues involved in the formation of the Fab–RBP complex is smaller than that of interacting residues in the RBP– TTR complex (Table 2) but the surface buried upon complex formation is comparable to that for the RBP– ˚ TTR complex (1588 A2) The contacts between interacting surfaces of RBP and Fab are shown in Fig 1C Mutational analysis of the RBP-binding determinants of TTR The amino acid sequences of RBP and TTR from different vertebrate species showing the residues of human RBP and TTR that are mainly involved in interactions, according to our structure of the TTR– RBP–Fab complex, are shown in Fig The functional and structural consequences of several TTR point mutations on the RBP–TTR interactions have been investigated The values of the dissociation constants for several complexes between human TTR variants and human RBP, as determined by means of fluorescence anisotropy titrations (Fig 3A), are reported in Table For some TTR mutations that abolish or weaken significantly the RBP–TTR interactions, the crystal structures of the variants have been determined or already available structures have been examined to provide details of the interference with protein–protein recognition by mutations V20S TTR variant Two Val20 residues present in two distinct TTR subunits (B and C, according to our designation of TTR subunits) are located at the center of a large hydrophobic patch in the contact area between TTR and RBP, so that their replacement by a hydrophilic residue is expected to significantly impair protein–protein interactions Accordingly, the V20S mutation has been found to almost abolish the binding affinity between RBP and TTR (Table and Fig 3A) To investigate the structural consequences of the V20S replacement on the RBP–TTR recognition, we have determined the ˚ 1.6 A resolution crystal structure of the V20S TTR variant The overall structure is very similar to that of the wild-type protein (PDB: 1F41) [25]: a superposition of equivalent Ca atoms gives an rmsd of 0.40 and ˚ 0.68 A for subunits A and B, respectively However, FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS 5843 Transthyretin–retinol-binding protein interactions G Zanotti et al A B C Fig Structure of the TTR–RBP–Fab complex (A) Representation of the overall structure of the TTR–RBP–Fab complex TTR: chain A, red; chain B, green; chain C, magenta; chain D, orange RBP: chains E and F, yellow Fab: heavy chains H and N, cyan; light chains L and M, blue (B) Detail of the contact between the TTR subunits A, B and C and the RBP molecule E [left side, color codes as in (A)] Center and right drawings show the interacting surfaces of RBP (center) and of the TTR subunits A, B and C (right) rotated by approximately 90° counterclockwise and clockwise, respectively, compared to the previous view It is possible to appreciate how a protuberance formed by loops 63–67 and 92–98 on the RBP surface fits into a crevice formed by the arrangement of three TTR subunits (C) Detail of the contact between the RBP molecule F and the Fab chains H and L [left side, color codes as in (A)] Center and right drawings show the interacting surfaces of RBP (center) and Fab (right), rotated through a vertical axis by approximately 90° counterclockwise and clockwise, respectively, compared to the previous view Residues 163–169 and the region preceding the a-helix contribute to the formation of the RBP epitope 5844 FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS G Zanotti et al Transthyretin–retinol-binding protein interactions Table Contacts between amino acid residues in the RBP–TTR– ˚ Fab complex characterized by interatomic distances within 4.0 A: interactions between RBP and TTR Interactions were analyzed using the program CONTACT of the CCP4 package [54] RBP chain E TTR chain A W91 V93 S95 K99 L35 L63 L64 S95 F96 L97 Q98 K99 W67 Retinol TTR chain B S100 R103a V122 D99a, S100a L82, G83 L82, G83, I84 L82a, R21 Y114b I84, S85b, Y114a S85a S85a S85b TTR chain C R21 G83, I84 G83b a Denotes the participation of TTR residues in at least one polar contact b An H-bond distance is present between at least two atoms of interacting residues of RBP and TTR significant differences are observed for loop region 98–103, which connects b-strands G to F and is flexible in most TTR structures, and for loop 19–22, which connects b-strands A and B and hosts the mutation The movement of the latter loop is not large (the Ca ˚ atom of Val20 is displaced by 1.6 A from its position in the wild-type structure) but the entire loop 19–22 is ˚ displaced by more than 1.5 A from its original position (Fig 3B), so that the change in the positions of Val20 and Arg21 of two distinct TTR subunits (Table 1) may interfere simultaneously with two interactions established with RBP Table Contacts between amino acid residues in the RBP–TTR– ˚ Fab complex characterized by interatomic distances within 4.0 A: interactions between RBP and Fab Interactions were analyzed using the program CONTACT of the CCP4 package [54] RBP chain F R 163 Q164 Y165 R166 Q149 K150 Q156 R163 L167 a Fab chain L a Fab chain H a F36, S95 , Y100 N34a, R54b R54b Y53, R54a, N57a Y103 F101, Y52, T30b, S31b D55b D102a D102b,Y103a Y103b An H-bond distance is present between at least two atoms of interacting residues of RBP and Fab b Denotes the participation of Fab residues in at least one polar contact I84S and I84A TTR variants A drastic effect on the recognition between TTR and RBP is caused by mutations at position 84 The in vitro binding of the I84S and I84A TTR variants to RBP is abolished or becomes almost negligible, respectively [37] (Table and Fig 3A) Accordingly, the lack of interaction between RBP and the amyloidogenic I84S TTR is known to lead to a markedly lowered plasma concentrations of RBP in individuals carrying the mutation due to the impaired transport function of the TTR variant [38] The explanation for this effect is not straightforward because the structures of the I84S (PDB: 2G4G) [39] and I84A (PDB: 2G4E) [39] TTR variants at neutral pH are very similar to that of the wild-type protein In both cases, the amino acid replacements are not associated with significant local conformational changes Ile84 occupies a central position in a hydrophobic patch of TTR involved in hydrophobic interactions with RBP; its replacement by a serine can perturb the polarity of the microenvironment at the interface, thereby impairing protein–protein recognition A reduction of the steric hindrance of the side chain of Ile84 as a consequence of the I84A mutation can instead perturb the interaction It is concluded that the residue at position 84 is particularly relevant for protein–protein recognition In this respect, it should be noted that, as in the case of Val20 and Arg21, two lle84 residues of two subunits present in two different dimers of the TTR tetramer are involved at the same time in the interactions with one RBP molecule (Table 1) Therefore, an amino acid substitution at position 84 in TTR can lead to the loss of two relevant contacts between two residues of an RBP molecule and both dimers of the TTR counterpart Moreover, we have demonstrated that, at variance with wild-type TTR, at pH 4.6, both I84S and I84A mutations induce a remarkable conformational change in the region comprising residue 84, with the disruption of the a-helix itself [39] It is tempting to speculate that the two amino acid replacements cause a destabilization of the TTR region hosting the mutation, as revealed by the structural alteration at acidic pH, which in turn may affect the interaction with RBP S85A TTR variant Ser85 appears to be relevant for the TTR–RBP interaction on the basis of the structure of the complex because it participates in several contacts with RBP residues: two are H-bond interactions, one between the Ser85 -OH group and the amide nitrogen of Lys99 of RBP and the other between the amide nitrogen of FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS 5845 Transthyretin–retinol-binding protein interactions G Zanotti et al A B Fig Multiple sequence alignments for RBP (A) and TTR (B) from different vertebrate species The residues that are identical in all the sequences considered for each alignment are shaded in red; the residues that are identical or chemically similar in at least five sequences for each alignment are denoted by red characters (similarity groups are: HKR, DE, STNQ, AVLIM, FYW, PG, C) The amino acid residues in the human TTR and RBP sequences more directly involved in RBP–TTR interactions (Table 1) are denoted by arrowheads Numbering and secondary structure elements are based on the structures of human RBP (PDB: 1JYD) and TTR (PDB: 1F41) GenBank or SwissProt accession numbers are: human TTR, PO2766; rat TTR, NP_036813; chicken TTR, CAA43000; zebrafish TTR, AAH81488; sea bream TTR, AF059193; trout TTR, CB497711 (EST sequence); human RBP, P02753; rat RBP, NM_013162; chicken RBP, P41263; zebrafish RBP, EF373650; sea bream RBP, AAF79021; trout RBP, P24774 Sequences containing the signal peptide have been reported when the N-termini of the mature proteins are not known Sequence alignments were constructed by CLUSTALW [58] and rendered with ESPRIPT [59] Ser85 and the carbonyl oxygen of Phe96 of RBP (Table 1) Although the latter can be preserved in the S85A variant, the former is lost, possibly contributing 5846 to an approximately five-fold decrease in binding affinity caused by the mutation (Table 3) It can be speculated that the loss of interactions caused by the FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS G Zanotti et al Transthyretin–retinol-binding protein interactions D99A and S100E TTR variants A These are important mutations because each of them causes an approximately 20-fold decrease in binding affinity of TTR for RBP (Table and Fig 3A) Both replaced residues interact with Lys99 of RBP and, moreover, Ser100 is close to Trp91 (Table 1) Consequently, it is conceivable that the replacements of Asp99 by a hydrophobic residue and of Ser100 by a charged residue significantly perturb the interactions However, it is likely that the effects of the D99A and S100E mutations are less drastic compared to the I84S and V20S replacements because the interactions involving residues at positions 99 and 100 are present at the periphery of a contact area between RBP and TTR Moreover, the new electrostatic situation for the TTR variants could result in new interactions of the mutated residues with the solvent B Y114F and Y114H TTR variants Fig Human TTR mutations affecting TTR–RBP interactions (A) Typical fluorescence anisotropy titrations of human holoRBP (3 lM) with human TTR: wild-type, black; V20S TTR, gray; I84S TTR, red; D99A TTR, green; Y114H TTR, blue; Y114F TTR, orange Fluorescence anisotropy values are plotted as a function of human TTR molar concentration Lines represent theoretical binding curves (for details, see Experimental procedures) corresponding to dissociation constants of 0.34 lM for wild-type TTR, 5.99 lM for D99A TTR, 1.04 lM for Y114H TTR and 0.17 lM for Y114F TTR (B) Stereo view showing the superposition of the Ca chain traces of wildtype TTR (PDB: 1F41, red) and the V20S TTR variant (green) in the area around residue 20 The side chains of residues Ser20 or Val20 and Arg21 are shown mutation may be partially compensated by a novel hydrophobic interaction in a hydrophobic patch involving Ala85 A special case is represented by Tyr114: its substitution for a phenylalanine lowers the dissociation constant of the protein–protein complex by an approximate factor of two, whereas the dissociation constant is three-fold higher when Tyr114 is replaced by a histidine (Table and Fig 3A) We have also determined the crystal structure of the amyloidogenic Y114H variant: no significant conformational changes have been observed, and, in particular, the side chain of His114 maintains the same position and orientation compared to Tyr114 The same holds for the position and orientation of the Phe114 residue present in the structure of both chicken TTR [26], which interacts well with RBP [17,40], and piscine TTR [27] The -OH group of Tyr114 forms an H-bond with the -OH group of Ser95 of RBP Moreover, Tyr114 is in a hydrophobic patch of TTR, despite its proximity to the protein surface Its replacement by a phenylalanine is not drastic in terms of modification of the surface potential but it leads to the loss of an H-bond interaction It must be assumed that the loss of a H-bond interaction for the Y114F variant is compensated by some conformational rearrangements that result in stronger hydro- Table Dissociation constants of complexes between human holoRBP and human TTR variants as determined by means of fluorescence anisotropy titrations Data represent the average of at least three independent measurements TTR Wild-type V20Sa V30Ma,b L55Pa,b L58Hb T60Aa,b I84Sa,b,c I84Aa S85A D99Ac S100Ec Y114Ha,b Y114Fc KD (lM) 0.35 –d 0.22 0.66 0.31 0.43 –e –d 1.64 6.23 5.81 1.12 0.18 a The structure for the TTR variant is available: V20S and Y114H [present study]; V30M [55]; L55P, [56]; T60A, [57]; I84S and I84A, [39] Amyloidogenic TTR variant c Position affected by significant amino acid replacements in piscine TTR compared to human TTR (Fig 2B) d Almost negligible interaction e Lack of interaction b FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS 5847 Transthyretin–retinol-binding protein interactions G Zanotti et al phobic interactions, possibly explaining the higher affinity between RBP and this TTR variant The replacement of Tyr114 by a potentially charged histidine could have a marked effect on the surface potential; the finding that the Y114H mutation does not drastically impair the RBP–TTR interaction suggests that His114 is not protonated in the protein–protein complex Modeling of the interactions of piscine TTR and RBP with protein counterparts within the TTR–RBP–Fab complex The observation that human TTR and RBP are bound in the TTR–RBP–Fab complex without undergoing significant conformational changes compared to the uncomplexed proteins prompted us to assess the ability of the two proteins from fish, which are unable to interact with each other [27], to fit within the structure of the TTR–RBP–Fab complex by replacing the corresponding human proteins present in the complex The structure of sea bream (Sparus aurata) TTR is known [27], whereas no structure for a fish RBP is available to date Therefore, only a theoretical model for the structure of sea bream RBP could be obtained with the SwissModel server [41] The structure of sea bream TTR was superimposed on that of human TTR present in the TTR–RBP–Fab complex, giving rise to a hypothetical model of the mixed piscine TTR–human RBP complex (Fig 4) The most relevant difference can be observed for loop 98–102 of all the subunits of TTR, especially for subunits A and C, due to some remarkable mutations affecting interacting residues (Table and Fig 2B) The conformation of loop 80–85 of piscine TTR also changes slightly (Fig 4), possibly due to relevant mutations in this area (Table and Fig 2B) The same holds for a model in which the human RBP structure is also replaced by a theoretical sea bream RBP structure, giving rise to a hypothetical piscine RBP–TTR complex (Fig 4) Conformational differences for TTR along with point mutations that have no effect on TTR structure (Fig 2B) may account for the experimentally determined lack of binding affinity between piscine TTR and RBP, as well as between piscine TTR and human RBP [27] Only limited conformational differences between piscine and human RBP are found Accordingly, the degree of conservation of the putative interacting residues is remarkably higher in piscine RBP than in piscine TTR; the only significant amino acid difference in piscine RBP compared to human RBP is K99T (Fig 2A) These features may explain the existence of an affinity, albeit weak, between piscine RBP and human TTR [27,42] Discussion The human holoRBP–TTR complex is relatively weak, being characterized by a dissociation constant of approximately 0.35 lm (Table 3) It has been suggested that this feature may be correlated with the need for the presence in plasma of a small but significant amount of uncomplexed holoRBP, which can thus leave the circulation more easily to deliver the retinol to the target tissues [7] A limited number of residues and retinol itself are mainly responsible for the relatively weak RBP–TTR interaction An important role played by the retinol hydroxyl end group in the interaction is consistent with the low binding affinity of apoRBP compared to that of holoRBP for TTR [16,17] and with the drastic interference with the interaction between the two proteins by RBP-bound fenretinide, a retinoid that bears a bulky end group in place of the retinol hydroxyl group [43,44] Moreover, the conformational change affecting one of the loops Fig Modeling of piscine TTR and RBP within the TTR–RBP–Fab complex Stereo view showing interacting regions between RBP (magenta) and TTR (orange) in the human RBP–TTR complex bound to Fab, with a superimposed model of the piscine RBP–TTR complex (green) based on the structure of sea bream TTR (PDB: 1OO2) and on a hypothetical model of the piscine RBP structure The two regions of TTR that differ significantly in the structures (98–102 and 80–85) are labeled 5848 FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS G Zanotti et al surrounding the opening of the b-barrel (in particular, residues Leu35 and Phe36) in apo-RBP compared to holoRBP [30,31] is likely to contribute to the weakening of the interaction of apoRBP with TTR due to the involvement of such a loop in RBP–TTR recognition Despite a few exceptions, the substitutions of hydrophilic for hydrophobic side chains in TTR contact regions generally have a rather pronounced dissociating effect on the RBP–TTR complex, consistent with the important role played by interfacial apolar interactions In general, the changes in the TTR molecule induced by amyloidogenic mutations not interfere with the interactions between RBP and TTR, unless the mutations are located in contact areas The amyloidogenic mutations V30M, L55P, L58H and T60A have a limited effect on the binding affinity between RBP and TTR (Table 3), in accordance with the lack of the replaced residues in contact areas; moreover, it can be inferred that such amyloidogenic mutations not cause large conformational changes in the TTR molecule that might affect indirectly the RBP–TTR recognition Conversely, the amyloidogenic I84S mutation, which affects a residue that is crucial for protein–protein interactions, causes the lack of recognition between RBP and TTR and an altered plasma transport of RBP by TTR [37,38] It might be hypothesized that the ability of RBP to interact well with relevant amyloidogenic TTR variants, such as V30M, L55P, L58H and T60A, can protect them from amyloid aggregation However, it should be noted that the plasma concentration of TTR is significantly higher than that of RBP [7], so that a protective effect of RBP on TTR can only be limited Despite the high symmetry of TTR, which is a homotetramer with virtually four identical binding sites for RBP, a : TTR : RBP complex is believed to be present in plasma due to the excess of TTR over RBP [7] Binding data obtained in solution [17,37,45] and structural data [35,36] have shown that a maximum of two RBP molecules can be bound by one TTR tetramer The binding of two RBP molecules to an uncomplexed TTR tetramer partially hinders the potential binding of two nearby RBP molecules, thereby limiting the possible interactions with tetrameric TTR to two RBP molecules [35,36] However, two distinct macromolecular organizations, both accounting for the : TTR : RBP stoichiometry, have been described for the heterologous and the homologous RBP–TTR complexes [35,36] The crystal structure of the TTR tetramer is characterized by 222 symmetry One of the three orthogonal two-fold axes runs through the central channel harboring the two thyroid hormone binding sites In the heterologous chicken RBP–human Transthyretin–retinol-binding protein interactions TTR complex, the two TTR-bound RBP molecules are related by one of the two available two-fold axes that are perpendicular to the central channel of TTR [35] Instead, in the homologous human RBP–human TTR complex, as well as in the case of our structure of the TTR–RBP–Fab complex, the two TTR-bound RBP molecules are related by the two-fold axis running through the central channel of TTR [36] (Fig 1A) Because the two situations are chemically equivalent, a possible explanation for the observed different assembly is that, in solution, both modes of assembly can be present and that the crystallization process selects one of them according to the best packing By comparing the structure of the human TTR– human RBP complex bound to Fab with those of the heterologous human TTR–chicken RBP complex [35] and of the homologous human TTR–human RBP complex [36], a good correspondence between these structures with regard to interacting surfaces of TTR and RBP has been found In the case of the human TTR–human RBP complex, however, it should be noted that one of the two TTR-bound RBP molecules has been reported to participate in the interaction with the last C-terminal amino acid residues (especially Leu182 and Leu183), thereby generating an asymmetry within the complex [36] At variance with this observation, our TTR–RBP–Fab structure does not reveal the presence of interactions between the carboxy terminus of RBP and TTR On the other hand, it should be noted that chicken RBP, in which eight C-terminal residues are missing compared to human RBP (Fig 2A), binds to human TTR with an affinity similar to that exhibited by human RBP [40; C Folli and R Berni, unpublished data], which suggests that the carboxy terminus of human RBP is not so crucial for the interaction with TTR The RBP–TTR complex is normally isolated from the serum of terrestrial vertebrates, such as mammals and birds [7] Moreover, purified human and chicken RBP and TTR have been found to cross-interact [40] By contrast, RBP could be isolated from the serum of different fish species only as uncomplexed protein [27,42,46], suggesting that, in fish, it is present in the circulation as an uncomplexed protein without affinity for TTR In accordance with this observation, the lack of binding affinity between purified piscine RBP and TTR has been established [27] The comparison of the amino acid sequences of piscine RBPs and TTRs with those of the same proteins from terrestrial vertebrates reveals the presence of remarkable differences in regions involved in protein–protein interactions for TTR, whereas only limited differences are present in the case of RBP (Fig 2A,B) In particular, the amino FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS 5849 Transthyretin–retinol-binding protein interactions G Zanotti et al acid replacements at positions 82, 84, 99 and 100 in piscine TTR compared to the human and chicken proteins are drastic and are present at positions critical for the interaction between RBP and TTR (Table and Fig 2B), and some of them (I84S, D99A and S100E) are shown in the present study to impair or abolish protein–protein recognition (Table and Fig 3A) The results obtained are consistent with the notion that evolutionary changes affecting a limited number of surface-exposed residues led to the appearance in terrestrial vertebrates of the TTR function of cotransport of retinol through the interaction with holoRBP in plasma, in addition to that of the distribution of thyroid hormones in the extracellular fluids Experimental procedures Materials HoloRBP was purified from human plasma as reported previously [17] Recombinant wild-type human TTR and TTR variants I84S and I84A were prepared and quantified as described previously [39] All chemicals were of analytical grade Site-directed mutagenesis, bacterial expression and purification of human TTR variants The recombinant human TTR variants V20S, L55P, L58H, T60A, S85A, D99A, S100E, Y114F and Y114H were prepared by PCR using the plasmid pET11b-human TTR [39] as template, a high-fidelity thermostable DNA polymerase (Pfu Ultra II Fusion HS DNA polymerase; Stratagene, La Jolla, CA, USA) and mutagenic primers complementary to opposite strands For each mutation, the product of reaction was treated with DpnI (New England Biolabs, Beverly, MA, USA) to digest the parental DNA template This procedure allowed us to select the newly synthesized and potentially mutated plasmids The products of each digestion were used to transform Escherichia coli XL1 Blue cells Single clones were then sequenced to confirm the occurrence of the desired mutation Finally, mutant plasmids were electroporated into E coli BL21 (DE3) cells The expression of TTR variants was induced by mm isopropyl thio-b-d-galactoside and, after incubation for h at 30 °C, cells were disrupted by sonication TTR variants were purified as described for wild-type TTR [39] Preamplification System (Gibco, Gaithersburg, MD, USA) The cDNA sequences encoding for the variable domains of the H and L chains of the monoclonal antibody A8P3 were PCR amplified using a mixture of 18 5¢ primer VKBACK mix and a mixture of five 3¢ primer VKFOR for the VK gene and 20 5¢ primer VHBACK mix and a mixture of five 3¢ primer VHFOR mix for the VH gene [48] Each domain was cloned using the TA cloning kit (Invitrogen, Carlsbad, CA, USA) and sequenced using an automated model 377 sequenator (Applied Biosystems, Foster City, CA, USA) The amino acid sequences of the variable domains of L and H chains of the antibody A8P3 are provided in Fig S1 Binding assay for the interaction between holoRBP and TTR variants To study the in vitro interaction of holoRBP with TTR variants, the highly fluorescent RBP-bound retinol provides an intense signal which is suitable for fluorescence polarization measurements [40] The intensities of the vertical (I ||) and horizontal (I^) components of the fluorescence of RBP-bound retinol (excitation at 330 nm and emission at 460 nm) were recorded at an angle of 90° to the vertically polarized excitation beam A correction factor, G, equal to I ¢^ ⁄ I ¢|| (where the primes indicate excitation polarized in a perpendicular direction) was used to correct for the unequal transmission of differently polarized light Fluorescence anisotropy (A) was determined according to the equation: A = (I || ) GI^) ⁄ (I || + 2GI^) Human holoRBP (0.7– 3.0 lm) in 0.05 m sodium phosphate (pH 7.2) and 0.15 m NaCl, at 20 °C, was titrated by adding aliquots of concentrated solutions of human TTR (wild-type or mutant forms) to the RBP-containing cuvette and the increase in fluorescence anisotropy of the RBP-bound retinol upon complex formation was monitored The fraction of RBP bound by TTR (a) was calculated for every point of the titration curves using the equation: a = (A ) Ao) ⁄ (Amax ) Ao), where A represents the fluorescence anisotropy value of RBP-bound retinol for a certain molar concentration of TTR, and Amax and Ao are the two limiting anisotropy values (i.e in the presence of an excess saturating TTR and in the absence of TTR, respectively) Binding data were analyzed as described [12] Fluorescence anisotropy measurements were carried out with a LS-50B spectrofluorometer (Perkin-Elmer, Waltham, MA, USA) Determination of the amino acid sequences of anti-RBP Fab variable domains Crystallization, data collection, structure determination and refinement for the TTR–RBP–Fab complex and the V20S and Y114 TTR variants Total RNA obtained from the cell line producing the antiRBP murine monoclonal antibody A8P3 [47] was subjected to retrotranscription into cDNA employing the Superscript Crystallization and preliminary X-ray data for the macromolecular complex formed by human transthyretin, human holoRBP and a murine anti-RBP Fab have been reported 5850 FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS G Zanotti et al Transthyretin–retinol-binding protein interactions Table Data collection Values in parentheses are for the outer resolution shell TTR–RBP–Fab complex Space group and cell parameters ˚ Resolution (A) Independent reflections Multiplicity Completeness (%) Rmergea a V20S TTR variant Y114H TTR variant C222, a = 159.62, b = 223.18, c = 121.43 15.72–3.36 (3.51–3.36) 25746 (1622) 3.0 (2.6) 84.4 (77.7) 3.3 (1.9) 0.16 (0.34) P21212, a = 42.00 b = 83.81, c = 65.58 65.5–1.59 (1.63–1.59) 30276 (2071) 3.2 (2.0) 99.1 (92.9) 17.3 (2.1) 0.051 (0.423) P21212, a = 42.56 b = 86.28, c = 64.83 64.8–2.30 (2.42–2.30) 9936 (1599) 5.9 (6.2) 99.3 (100) 5.2 (3.9) 0.095 (0.156) hkl À Rmerge ¼ Rhkl jIRIhklhIhkl ij previously [49] The structure has been solved by molecular replacement, using the models of the single components of the complex as templates (for RBP, PDB: 1RBP; for TTR, PDB: 1F41; for Fab, a model was obtained with the server EXPASY) [41] The crystallographic refinement was carried on using the software cns [50], imposing noncrystallographic symmetry restraints throughout all cycles Three hundred and thirty-four water molecules were added in the last cycles of the refinement The addition of these water molecules caused 0.02 and 0.01 unit reductions, respectively, of R and Rfree factors A relatively high R factor of 0.239, with an Rfree of 0.312, for the final model of the TTR–RBP–Fab complex is justified by the quite small size of the crystals and the large dimensions of the asymmetric unit, which contains one molecule of the complex, accounting for a total of 10 polypeptide chains (one TTR tetramer, two RBP and two Fab) and a molecular mass of approximately 200 kDa However, the stereochemical parameters for the RBP and TTR components of the complex are quite good for this resolution Moreover, the Rfree for the TTR– Table Refinement statistics Values in parentheses are for the outer resolution shell TTR–RBP–Fab complex Protein atoms Solvent molecules ⁄ ligand atoms Rcryst.a Rfree ˚ Mean B value (A2) Ligands mean B ˚ value (A2) rmsd from ideal values ˚ Bond lengths (A) Bond angles (°) V20S TTR variant Y114H TTR variant 13 058 334 ⁄ 42 1765 165 ⁄ 1783 94 0.239 (0.313) 0.312 (0.357) 37.5 13.4 0.209 (0.277) 0.229 (0.303) 17.8 – 0.218 (0.258) 0.281 (0.326) 23.3 – 0.010 1.5 0.011 1.29 0.006 1.3 jj Rcryst ¼ Rhkl RFo jÀkjjFc jj, where |Fo| and |Fc| are the observed and calcuhkl jFo lated structure factor amplitudes for reflection hkl, applied to the work (Rcryst) and test (Rfree) (7% omitted from refinement) sets, respectively a RBP–Fab complex (0.312) appears to be significantly better than that (0.403) obtained for the crystals of the human TTR–BRP complex [36] Crystals of the V20S TTR variant were obtained at 295 K by the vapor diffusion method, using 0.066 m CaCl2, 19% (w ⁄ v) poly(ethyelene glycol)-400 and 0.13 m sodium Hepes (pH 7.5) as precipitant reservoir solution Crystals of the Y114H variant were grown at pH 5.6 in 100 mm Na citrate buffer, using m ammonium sulfate as precipitant The crystals were frozen at 100 K and the data collected at the X-ray diffraction beam-line ID29 of the ESRF synchrotron (Grenoble, France) Because the crystals were isomorphous with the wild-type protein, the molecular model of the latter (PDB: 1F41) was subjected to a rigid-body refinement, followed by some cycles of restrained least-squares with the software refmac [51] or cns [50] and by visual inspection and manual rebuilding with the program coot [52] The R factor for the final model is 0.209 (with an Rfree of 0.229) for the V20S TTR variant and 0.218 (and an Rfree of 0.281) for Y114H TTR variant Both models present a good stereochemistry, as assessed by the program procheck [53] The overall final statistics for the structures of the TTR–RBP– Fab complex and the V20S and Y114H TTR variants are provided in Tables and Calculations of the areas buried following complex formation were performed using ˚ the software areaimol with a probe radius of 1.4 A [54] Acknowledgements The technical assistance of the staff of beamlines ID29 (ESRF, Grenoble) and XRD1 (ELETTRA, Trieste) with respect to data collection for the V20S and Y114H TTR variants and the TTR–RBP–Fab complex, respectively, is gratefully acknowledged Vaida Arcisauskaite took part, as an undergraduate student, in the refinement of the structure of the V20S TTR variant We thank Riccardo Percudani for fruitful discussions This study was supported by PRIN Projects ` of the ‘Ministero dell’Universita e della Ricerca’ (Rome, Italy) and by the Universities of Parma and Padua, 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The following supplementary material is available: Fig S1 Amino acid sequences of the variable domains of the H and L chains of the anti-RBP monoclonal antibody A8P3 This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary material supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS ... b-strands H and F To form the tetramer, two dimers associate back to back, mainly through hydrophobic contacts between residues of the loops formed by b-strands A and B and b-strands G and H One of. .. crucial for protein? ? ?protein interactions, causes the lack of recognition between RBP and TTR and an altered plasma transport of RBP by TTR [37,38] It might be hypothesized that the ability of RBP... interaction between RBP and TTR (Table and Fig 2B), and some of them (I84S, D99A and S100E) are shown in the present study to impair or abolish protein? ? ?protein recognition (Table and Fig 3A)