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The transthyretin-retinol-binding protein complex
Biochimica et Biophysica Acta 1482 (2000) 65^72 www.elsevier.com/locate/bba
Review
The transthyretin-retinol-binding protein complex Hugo L. Monaco Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy Received 4 October 1999; received in revised form 26 November 1999; accepted 1 December 1999
Abstract Transthyretin (TTR, formerly called prealbumin), one of the transporters of the hormone thyroxine and the lipocalin retinol-binding protein (RBP), the specific carrier of the vitamin, are known to form, under physiological conditions, a macromolecular complex that is believed to play an important physiological role: prevention of glomerular filtration of the low molecular weight RBP in the kidneys. The physiological significance of complex formation is discussed first, followed by a brief description of the three-dimensional structure of the two participating proteins. The two X-ray models of the complex available are subsequently discussed and compared and finally the non-crystallographic evidence that supports these models is reviewed. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Transthyretin; Prealbumin; Retinol-binding protein; Macromolecular complex; Protein-protein interaction; Crystal structure
1. The complex: physiological role Retinol transport in the plasma of vertebrates from the liver, which is the body's major storage organ, to the peripheral tissues takes place with the vitamin bound to a single speci¢c protein: the lipocalin retinol-binding protein [1] (RBP, see previous review). RBP circulates in plasma forming a macromolecular complex with another transport protein, the carrier of thyroxine transthyretin (TTR), formerly called prealbumin (for a comprehensive review see [2]). Both apo and holo RBP can form the complex with TTR but the dissociation constant of the former is signi¢cantly higher [3] which is consistent with a vitamin delivery mechanism in which the stable retinol-containing complex is retained in plasma while the single low molecular weight apo RBP is cleared from the circulation by glomerular ¢ltration [4]. Binding to TTR (a tetramer of total MW = 54 000) serves thus to prevent the loss of both the low molecular weight RBP (MW = 21 000)
and of its bound retinol. One of the major sites of synthesis of the two members of the macromolecular complex is the liver [5,6] and secretion of RBP has been shown to be regulated by retinol since in its absence apo RBP accumulates in the endoplasmic reticulum of the hepatocyte wherefrom it is quickly released as soon as the cellular level of retinol increases [7]. Although it was initially proposed that the complex formed in plasma from the two independently secreted proteins [8], more recent evidence indicates that the complex is formed in the hepatocytes [9,10] and that in its formation the participation of a chaperone is likely [11]. It has been reported that vitamin A depletion has no e¡ect on the liver concentration of TTR [12], but the evidence that supports intracellular complex formation is convincing enough to let us suspect that the two proteins that interact in this complex are probably more intimately related than it had been thought. Though the exact mechanism of vitamin delivery is still a matter of debate [13], evidence for the presence of
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cell surface receptors has been presented [14^17]. The kinetics of the process leading to the transfer of retinol to the target cells has shown that the vitamin is transferred from the complex so that there is not a previous dissociation step of the two proteins which instead separate from one another after the loss of the vitamin [18]. The fact that in rat hepatocytes the amount of vitamin taken up from the complex is roughly twofold greater than that taken up from holo RBP has raised the interesting possibility that TTR may also participate in the interactions with the putative receptors [19]. The normal RBP concentration in human plasma is about 2 WM [4] and that of TTR about 4.5 WM [2]. Therefore the stoichiometry of the complex in vivo is believed to be of one RBP molecule per TTR tetramer. However, when the complex was formed in vitro adding the appropriate amounts of the two proteins, other stoichiometries were found [20,21], and there is con£icting evidence in the earlier literature as to the maximum number of RBP molecules that can be bound by a TTR tetramer [22]. Nevertheless, the crystallographic results discussed below establish unequivocally that the number of RBP molecules bound by a TTR tetramer cannot be higher than two. The presence of TTR has been demonstrated in more than 15 vertebrate species [23] and more than 20 amino acid sequences have been determined either by chemical or genome sequencing. In particular, it is interesting to note the presence of a TTR-like protein precursor in the genome of Bacillus subtilis (gene yunM) and Escherichia coli (gene yedX) [24]. In mammals, the sequence similarities and quaternary structure of the molecule are strikingly preserved and the percentage identity in amino acid sequence between di¡erent species is 87% [25^27]. TTR is thus one of the most strongly conserved plasma proteins. Equally well preserved appear to be the properties of RBP and though fewer sequences are known, the conservation of similarities appears to be as high as in the case of TTR [28]. The complex has been studied in several di¡erent organisms and cross-reactivity among RBP and TTR from di¡erent species, some times quite distant in evolution, was demonstrated many years ago [29]. Two examples may be mentioned: the complex human TTR-chicken RBP [30] and the complex human TTR-trout RBP [28].
Although the a¤nity constant of the two proteins for each other can vary somewhat depending on the exact conditions of measurement, e.g. pH, ionic strength and species, most determinations have given dissociation constants of the order of 1036 ^1037 M [18]. The values are found to be quite comparable among the homologous complexes belonging to different species and are also very similar for the chimeric complexes formed by RBP and TTR from different origin. The interactions of the two proteins in the complex have also been examined using mass spectrometry [31] which con¢rmed that the stoichiometry is of a maximum of two RBP molecules per TTR tetramer. In addition, these experiments furnished estimates for the solution dissociation constants which were found to be 1.5U1037 M for the ¢rst RBP molecule bound and 3.5U1035 M for the second. This result supports an earlier proposal that binding of the ¢rst RBP molecule induces cooperative e¡ects that decrease the a¤nity of TTR for the second RBP molecule [30]. The ligand transported by the complex is exclusively all-trans retinol though the a¤nity of RBP for other retinoids, most notably retinoic acid, is quite similar [32]. This transport selectivity arises not from the binding of the ligands to RBP but from the formation of the macromolecular complex with TTR. It has been shown that the recognition between the two members of the protein complex is correlated with the nature of the retinoid liganded to RBP and that RBP bound to the retinoid fenretinide, which exposes on the protein surface the bulky hydroxyphenyl group, has a negligible a¤nity for TTR [33]. 2. Structure of the two members of the protein complex The three-dimensional structure of human [34^36] and bovine [37] holo and apo RBP as well as the higher resolution structure of porcine RBP [38] have been extensively described (see also previous review). The wild type [39^41] and several mutants of human TTR [42^44] and of complexes of the wild type with pharmacologically important compounds [45^47] as well as chicken TTR [48] have been the subject of very detailed X-ray structural studies. The
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Fig. 1. Ribbon representation of the structure of the hexameric complex (RBP)2 TTR determined by X-ray analysis of the chimeric crystals grown using human TTR and chicken RBP. The retinol molecules are represented as CPK models. This view is looking down the z axis of the TTR tetramer as de¢ned by Blake et al. [39]. The x and y axes are in the plane of the ¢gure, the ¢rst is horizontal and the second vertical. They intercept in the center of the TTR channel, the hormone-binding site, which is represented empty in the ¢gure.
two members of the complex are thus structurally very well characterized. TTR, the ¢rst plasma protein whose structure was determined by X-ray di¡raction studies, is a tetramer of four identical subunits, each 127 amino acids long (see Fig. 1). In the tetramer, the four monomers do not occupy equivalent positions but are organized as a dimer of dimers. Each monomer presents two extensive L sheets, each composed of four strands that are all antiparallel with one exception [39^41]. Two monomers form then a very stable dimer by extending their two L sheets through hydrogen bonding that involves the four strands (two from each monomer) at the edges of the two subunits. The two dimers of the tetramer are separated by a channel and in contact through î in symmetry related loops. The channel, about 10 A diameter, has been shown to be the ligand-binding site [39]. The three orthogonal molecular twofold axes of TTR have been designated x, y and z [39], the latter being coincident with the ligand binding channel of the molecule. The RBP molecule is shaped like a calyx made up of eight strands of antiparallel L sheet which are
followed topologically by a short alpha helical segment ([35]; see also previous review). Into this calyx, the retinol molecule binds with the L ionone ring buried deepest and with the alcohol moiety pointing to the outside on the surface of the molecule. Recall that the presence of the vitamin bound to RBP is correlated to the formation of a more stable complex with TTR [33]. Well re¢ned models of RBP liganded to several di¡erent retinoids are also available [49]. Though it was initially thought that removal of the retinol molecule from the calyx would result in major conformational changes, X-ray crystallographic studies of human and bovine RBP [36,37] have shown that the transition from holo- to apoprotein involves only very subtle modi¢cations. The most important is a conformational change on the loop extending from amino acids 34 to 37, in particular, Leu 35 and Phe 36. The space left empty by the removal of the vitamin is ¢lled in both cases by the aromatic ring of Phe 36 and solvent molecules and the movement of the Phe side chain drags the nearby amino acids into positions which are di¡erent from those adopted in the holoprotein.
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3. Crystal structures of the complex Two models of the TTR-RBP complex have been determined from X-ray di¡raction data which extended in both cases to moderate resolution (3.1 î respectively). The two structures deterand 3.2 A mined are that of the chimeric complex humanTTR-chicken-RBP [50] and that of the homologous complex human-TTR-human-RBP [51]. For the chimeric complex it was found that the imposition of a stoichiometry of two RBP molecules per TTR tetramer was essential for successful crystallization [52]. An even higher ratio of 2.5^3 RBP molecules per TTR tetramer was used for the crystallization of the human homologous complex. In both cases the species found in the crystals was the hexamer TTR(RBP)2 but the quaternary structure of the complex in the two cases turned out to be di¡erent. Fig. 1 is a ribbon representation of the chimeric hexamer in which the view is looking down the z axis of the TTR tetramer as de¢ned by Blake et al. [39]. The x and y axes are in the plane of the ¢gure. The x axis is horizontal and separates the two TTR dimers that assembled constitute the tetramer. The y axis is vertical and in the chimeric complex it is the twofold axis that relates the two RBP molecules as should be apparent from the ¢gure. Thus, in this complex the
two RBP molecules are found to interact most extensively with the same TTR dimer. In the homologous human complex, the twofold axis which is preserved is the z axis instead and so the two RBP molecules are found to interact most extensively with the opposite dimers of the TTR tetramer. The reason why two di¡erent twofold axes are preserved in the two crystals studied is not clear but at this point it is probably pertinent to emphasize that the molecular species we have to imagine circulating in plasma is the pentamer and not the hexamer which was created arti¢cially in vitro in both cases and which had to have a higher symmetry to facilitate the packing of the complexes in the crystal lattices. In both the chimeric and the human homologous complex, the structure of the hexamer precludes the possibility of the binding of a third and fourth RBP molecules. In the chimeric hexamer in which the y axis is the preserved twofold, the two RBP molecules bind to the TTR tetramer along an axis which is parallel to the x axis (Fig. 1) and which is su¤ciently close to it to hinder the potential binding of the two other RBP molecules that would be required to satisfy this symmetry element in an octamer. Something analogous happens in the homologous human hexamer in which the two RBP molecules bind on the opposite sides of the x axis but again too close to it
Table 1 Signi¢cant contacts in the TTR-RBP complex in the two structures determined by X-ray crystallography RBP
Distance chimeric complex î) (A
Distance homologous complex î) (A
TTR
E E E E E E E E E E E E E E E
3.41 4.20 3.45 2.73 4.54 4.07 3.09 2.69 3.38 3.37 3.44 2.80 2.70 3.87 3.15
4.24 5.03 4.04 3.57 4.92 3.68 2.71 3.25 3.46 3.30 3.25 3.59 5.47 4.33 3.48
B (cc) D C (cc) A C (cc) A A (cc) C A (cc) C A (cc) C B (cc) D B (cc) D B (cc) D B (cc) D C (cc) A B (cc) D A (cc) C B (cc) D B (cc) D
^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
35 (Leu) [CD2] 67 (Trp) [CZ2] 67 (Trp) [CD2] 89 (Lys) [NZ] 89 (Lys) [NZ] 91 (Trp) [NE1] 95 (Ser) [OG] 96 (Phe) [CO] 96 (Phe) [N] 96 (Phe) [CB] 96 (Phe) [CE1] 97 (Leu) [CO] 99 (Lys) [NZ] 99 (Lys) [N] (Retinol) [OH]
(hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc)
^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
83 (Gly) [CA] 20 (Val) [CG1] 84 (Ile) [CD1] 99 (Asp) [CO] 99 (Asp) [OD2] 100 (Ser) [CO] 114 (Tyr) [OH] 85 (Ser) [N] 114 (Tyr) [OH] 84 (Ile) [CG2] 21 (Arg) [CG] 85 (Ser) [OG] 99 (Asp) [OD2] 85 (Ser) [OG] 83 (Gly) [CO]
The RBP molecule selected in both cases is chain E and it is in contact with the chains de¢ned A, B, and C in the TTR tetramer of the chimeric complex. In the homologous complex the three equivalent TTR monomers are de¢ned as D, C and A. The ¢rst column gives the distance in the chimeric complex [50] and the second the distance in the homologous human complex [51].
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Fig. 2. Schematic representation of the main protein-protein interactions found in the chimeric complex. An asterisk identi¢es Ile 84 which is present in the contact area of the di¡erent TTR monomers, B and C, that are part of the two separate dimers of the tetramer.
to allow for the formation of the octamer. Thus the two structures are consistent with a stoichiometry of a maximum of two RBP molecules per TTR tetramer. The main amino acids involved in the intermolecular contacts between an RBP molecule and the TTR tetramer, initially identi¢ed in the chimeric complex on the basis of a distance cuto¡ [50], are listed in Table 1, along with the distances measured in that model. Table 1 also gives the distances measured between the equivalent atoms in the homologous human complex [51]. From the table one can conclude that the two X-ray structures are substantially in agreement with one another but there is one major signi¢cant di¡erence between the two models. In the homologous human complex, the last ¢ve RBP amino acids, which are not present in chicken RBP (the
species used for the chimeric complex), are found to interact with the TTR tetramer in the case of one of the RBP molecules in the hexamer and not in the other. Thus, in the homologous complex, one of the RBP molecules interacts more extensively with the TTR tetramer than the other so that in fact the molecular twofold axis in the hexamer is not strictly preserved. The physiological signi¢cance of this additional interaction is discussed in [51]; the reasons for this asymmetry in the binding of the two RBP molecules in the case of the human homologous complex are not known. Fig. 2, taken from [50], is a cartoon representation of the most important interactions found in the chimeric complex between an RBP molecule and the TTR tetramer. Notice that the RBP molecule interacts with three di¡erent TTR monomers and that
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there is one amino acid, Ile 84, in two TTR monomers, one from each dimer, that participates in the interaction. In the homologous complex, two other amino acids belonging to the two di¡erent TTR dimers were identi¢ed as participating in the interaction: Val 20 and Arg 21 [51]. The RBP amino acids present in the protein-protein recognition area are located at the entrance of the L barrel and the retinol hydroxyl group has its oxygen at a distance of about î from the polypeptide chain of a TTR mono3^3.5 A mer. A fairly large hydrophobic area is situated between the B and C TTR monomers and the RBP E molecule in the chimeric complex. These hydrophobic contacts are among amino acids which are exposed to the solvent in the uncomplexed proteins and are therefore a very important driving force in complex formation. There are 27 amino acid di¡erences in the primary structure of chicken and human TTR and 23 di¡erences in the sequences of chicken and human RBP [53^56]. Of those amino acids, only one in the case of TTR, amino acid number 114 (a Tyr in human TTR and a Phe in the chicken counterpart) is found in the region of interprotein contacts of the chimeric complex. In the case of RBP it is also only one of the di¡erent amino acids which is found to participate in the intermolecular contacts in the homologous human complex [51]: amino acid number 64 which is a Leu in human RBP and a Phe in the chicken protein. This very high degree of conservation in the regions of protein-protein contacts in the complex emphasizes the physiological relevance of complex formation and its importance in animal physiology. 4. Experimental evidence that supports the X-ray models of the complex The two X-ray models described above are in agreement with the results of a series of experimental studies of which we will give a brief account here. It is known that the a¤nity of RBP for TTR is enhanced at high and reduced at low ionic strength [20] which is consistent with the participation of a hydrophobic area in the intermolecular contacts. Complex formation stabilizes the binding of retinol to RBP [57,58] and substitution of this ligand can
impair or even prevent the interaction of the two macromolecules [33,59] in agreement with the participation of the retinol hydroxyl group in the contacts with TTR. The binding of thyroxine to TTR is identical in the complex and in the isolated protein in accordance with the fact that the ligand binding site is located in an area that does not participate in any way in the macromolecular interactions [20,60]. Chemical modi¢cation of the tryptophan residues of RBP isolated and in the complex has shown that there is at least one Trp which reacts in the isolated protein and is instead protected upon complex formation, which implies that it is located in the area where the protein-protein contacts are established [61]. The identity of this residue was not established chemically but the two X-ray models of the complex discussed above have the same two Trp residues in the contact area: Trps 67 and 91. Acetylation of holo RBP with N-acetylimidazole (using conditions under which both Lys and Tyrs react) disrupts its binding to TTR and deacetylation of the tyrosines with hydroxylamine fails to produce RBP with a normal a¤nity for TTR [62]. This result indicates that there is at least a Lys and that there are no Tyrs present in the RBP area that participates in the protein-protein contacts. As Table 1 shows, the two models have Lys 89 and 99 in the surface in contact with TTR. The reduced a¤nity of apo RBP for TTR can also be explained by the two X-ray models. X-Ray structural studies had shown that in the holo to apo transition in RBP the only signi¢cant change involves amino acids 34^37 [36,37]. This area of the molecule, and in particular Leu 35, is present in the proteinprotein contact area in both X-ray models. Site directed mutagenesis studies on human RBP have con¢rmed the role of Leu 35 and also shown that in the double mutant in which Leu 63 and Leu 64 are changed to Arg and Ser the ability to bind to TTR is reduced [63]. Deletion of loop 92^98 resulted in complete loss of the ability to interact with TTR. Both X-ray models are consistent with the crucial role of this loop in the intermolecular interactions. More than 50 pathological variants of human TTR have been described [2,64]. They are mostly associated to the severe disease called familial amyloidotic polyneuropathy [2,42] but also to senile systemic amyloidosis [65] and euthyroid hyperthyroxine-
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mia [43]. According to the two X-ray models of the complex, in three of these mutants the residues altered [66^68] are involved in the interactions with RBP. In the ¢rst of this mutants, identi¢ed in Japan, the mutation changed Tyr 114 into a Cys [66] and a detailed characterization of the altered protein was not reported. The second mutant, in which Val 20 was substituted by an Ile, was associated with lateonset cardiac amyloidosis [67]. The plasma levels of RBP in this patient were not investigated. The third mutation, found in patients with the disease called Indiana type hereditary amyloidosis, has the substitution Ile 84CSer and the characteristic of showing markedly decreased levels in serum RBP [69]. A quantitative determination of the dissociation constant of the complex of this mutant with normal RBP showed that the a¤nity of the altered TTR for normal RBP is negligible, which explains the reduced RBP concentration found in the plasma of these patients [70]. In both X-ray models of the complex, this residue, Ile 84, is present twice at the interface with RBP and the monomers to which the residues belong are in di¡erent TTR dimers, which explains the dramatic e¡ect that this single mutation has on the stability of the complex. When the interactions of RBP with TTR were analyzed using mass spectrometry, the main conclusions reached were found to be in close agreement with the X-ray model of the chimeric complex [31]. References [1] M. Kanai, A. Raz, D.S. Goodman, J. Clin. Invest. 47 (1968) 2025^2044. [2] Y. Ingenbleek, V. Young, Annu. Rev. Nutr. 14 (1994) 495^ 533. î . Albertsson, B. Hansson, Eur. J. Biochem. 99 [3] G. Fex, P.-A (1979) 353^360. [4] D.S. Goodman, Plasma retinol-binding protein, in: M.B. Sporn, A.B. Roberts, D.S. Goodman (Eds.), The Retinoids, vol. 2, Academic Press, New York, 1984, pp. 41^88. [5] D.R. Soprano, K.J. Soprano, D.S. Goodman, J. Lipid Res. 27 (1986) 166^171. [6] P.W. Dickson, G.J. Howlett, G. Schreiber, J. Biol. Chem. 260 (1985) 8214^8219. ê brink, P.A. [7] H. Ronne, C. Ocklind, K. Wiman, L. Rask, B. O Peterson, J. Cell Biol. 96 (1983) 907^910. [8] M. Navab, J.E. Smith, D.S. Goodman, J. Biol. Chem. 252 (1977) 5107^5114.
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[51] H.M. Naylor, M.E. Newcomer, Biochemistry 38 (1999) 2647^2653. [52] H.L. Monaco, F. Mancia, M. Rizzi, A. Coda, J. Mol. Biol. 244 (1994) 110^113. [53] Y. Kanda, D.S. Goodman, R.E. Can¢eld, F.J. Morgan, J. Biol. Chem. 249 (1974) 6796^6805. [54] W. Duan, M.G. Achen, S.J. Richardson, M.C. Lawrence, R.E.H. Wettenhall, A. Jaworowski, G. Schreiber, Eur. J. Biochem. 200 (1991) 679^687. [55] L. Rask, H. Anundi, P.A. Peterson, FEBS Lett. 104 (1979) 55^58. [56] A.V. Vieira, K. Kuchler, W.J. Schneider, DNA Cell Biol. 14 (1995) 403^410. [57] P.A. Peterson, J. Biol. Chem. 246 (1971) 44^49. [58] D.S. Goodman, R.B. Leslie, Biochim. Biophys. Acta 260 (1972) 670^678. [59] J. Horwitz, J. Heller, J. Biol. Chem. 248 (1973) 6317^6324. [60] A. Raz, D.S. Goodman, J. Biol. Chem. 244 (1969) 3230^ 3237. [61] J. Horwitz, J. Heller, J. Biol. Chem. 249 (1974) 7181^7185. [62] J. Heller, J. Horwitz, J. Biol. Chem. 250 (1975) 3019^3023. [63] A. Sivaprasadarao, J.B. Findlay, Biochem. J. 300 (1994) 437^442. [64] M.J.M. Saraiva, Hum. Mutat. 5 (1995) 191^196. [65] D.R. Jacobson, P.D. Gorevic, J.N. Buxbaum, Am. J. Hum. Genet. 47 (1990) 127^136. [66] S. Ueno, T. Uemichi, S. Yorifuji, S. Tarui, Biochem. Biophys. Res. Commun. 169 (1990) 143^147. [67] D.R. Jacobson, T. Pan, R.A. Kyle, J.N. Buxbaum, Hum. Mutat. 9 (1997) 83^85. [68] M.R. Wallace, P.M. Conneally, M.D. Benson, Am. J. Hum. Genet. 43 (1988) 182^187. [69] M.D. Benson, F.E. Dwulet, Arthritis Rheum. 26 (1983) 1493^1498. [70] R. Berni, G. Malpeli, C. Folli, J.R. Murrell, J.J. Liepnieks, M.D. Benson, J. Biol. Chem. 269 (1994) 23395^23398.
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Review
The transthyretin-retinol-binding protein complex Hugo L. Monaco Biocrystallography Laboratory, Department of Science and Technology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy Received 4 October 1999; received in revised form 26 November 1999; accepted 1 December 1999
Abstract Transthyretin (TTR, formerly called prealbumin), one of the transporters of the hormone thyroxine and the lipocalin retinol-binding protein (RBP), the specific carrier of the vitamin, are known to form, under physiological conditions, a macromolecular complex that is believed to play an important physiological role: prevention of glomerular filtration of the low molecular weight RBP in the kidneys. The physiological significance of complex formation is discussed first, followed by a brief description of the three-dimensional structure of the two participating proteins. The two X-ray models of the complex available are subsequently discussed and compared and finally the non-crystallographic evidence that supports these models is reviewed. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Transthyretin; Prealbumin; Retinol-binding protein; Macromolecular complex; Protein-protein interaction; Crystal structure
1. The complex: physiological role Retinol transport in the plasma of vertebrates from the liver, which is the body's major storage organ, to the peripheral tissues takes place with the vitamin bound to a single speci¢c protein: the lipocalin retinol-binding protein [1] (RBP, see previous review). RBP circulates in plasma forming a macromolecular complex with another transport protein, the carrier of thyroxine transthyretin (TTR), formerly called prealbumin (for a comprehensive review see [2]). Both apo and holo RBP can form the complex with TTR but the dissociation constant of the former is signi¢cantly higher [3] which is consistent with a vitamin delivery mechanism in which the stable retinol-containing complex is retained in plasma while the single low molecular weight apo RBP is cleared from the circulation by glomerular ¢ltration [4]. Binding to TTR (a tetramer of total MW = 54 000) serves thus to prevent the loss of both the low molecular weight RBP (MW = 21 000)
and of its bound retinol. One of the major sites of synthesis of the two members of the macromolecular complex is the liver [5,6] and secretion of RBP has been shown to be regulated by retinol since in its absence apo RBP accumulates in the endoplasmic reticulum of the hepatocyte wherefrom it is quickly released as soon as the cellular level of retinol increases [7]. Although it was initially proposed that the complex formed in plasma from the two independently secreted proteins [8], more recent evidence indicates that the complex is formed in the hepatocytes [9,10] and that in its formation the participation of a chaperone is likely [11]. It has been reported that vitamin A depletion has no e¡ect on the liver concentration of TTR [12], but the evidence that supports intracellular complex formation is convincing enough to let us suspect that the two proteins that interact in this complex are probably more intimately related than it had been thought. Though the exact mechanism of vitamin delivery is still a matter of debate [13], evidence for the presence of
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cell surface receptors has been presented [14^17]. The kinetics of the process leading to the transfer of retinol to the target cells has shown that the vitamin is transferred from the complex so that there is not a previous dissociation step of the two proteins which instead separate from one another after the loss of the vitamin [18]. The fact that in rat hepatocytes the amount of vitamin taken up from the complex is roughly twofold greater than that taken up from holo RBP has raised the interesting possibility that TTR may also participate in the interactions with the putative receptors [19]. The normal RBP concentration in human plasma is about 2 WM [4] and that of TTR about 4.5 WM [2]. Therefore the stoichiometry of the complex in vivo is believed to be of one RBP molecule per TTR tetramer. However, when the complex was formed in vitro adding the appropriate amounts of the two proteins, other stoichiometries were found [20,21], and there is con£icting evidence in the earlier literature as to the maximum number of RBP molecules that can be bound by a TTR tetramer [22]. Nevertheless, the crystallographic results discussed below establish unequivocally that the number of RBP molecules bound by a TTR tetramer cannot be higher than two. The presence of TTR has been demonstrated in more than 15 vertebrate species [23] and more than 20 amino acid sequences have been determined either by chemical or genome sequencing. In particular, it is interesting to note the presence of a TTR-like protein precursor in the genome of Bacillus subtilis (gene yunM) and Escherichia coli (gene yedX) [24]. In mammals, the sequence similarities and quaternary structure of the molecule are strikingly preserved and the percentage identity in amino acid sequence between di¡erent species is 87% [25^27]. TTR is thus one of the most strongly conserved plasma proteins. Equally well preserved appear to be the properties of RBP and though fewer sequences are known, the conservation of similarities appears to be as high as in the case of TTR [28]. The complex has been studied in several di¡erent organisms and cross-reactivity among RBP and TTR from di¡erent species, some times quite distant in evolution, was demonstrated many years ago [29]. Two examples may be mentioned: the complex human TTR-chicken RBP [30] and the complex human TTR-trout RBP [28].
Although the a¤nity constant of the two proteins for each other can vary somewhat depending on the exact conditions of measurement, e.g. pH, ionic strength and species, most determinations have given dissociation constants of the order of 1036 ^1037 M [18]. The values are found to be quite comparable among the homologous complexes belonging to different species and are also very similar for the chimeric complexes formed by RBP and TTR from different origin. The interactions of the two proteins in the complex have also been examined using mass spectrometry [31] which con¢rmed that the stoichiometry is of a maximum of two RBP molecules per TTR tetramer. In addition, these experiments furnished estimates for the solution dissociation constants which were found to be 1.5U1037 M for the ¢rst RBP molecule bound and 3.5U1035 M for the second. This result supports an earlier proposal that binding of the ¢rst RBP molecule induces cooperative e¡ects that decrease the a¤nity of TTR for the second RBP molecule [30]. The ligand transported by the complex is exclusively all-trans retinol though the a¤nity of RBP for other retinoids, most notably retinoic acid, is quite similar [32]. This transport selectivity arises not from the binding of the ligands to RBP but from the formation of the macromolecular complex with TTR. It has been shown that the recognition between the two members of the protein complex is correlated with the nature of the retinoid liganded to RBP and that RBP bound to the retinoid fenretinide, which exposes on the protein surface the bulky hydroxyphenyl group, has a negligible a¤nity for TTR [33]. 2. Structure of the two members of the protein complex The three-dimensional structure of human [34^36] and bovine [37] holo and apo RBP as well as the higher resolution structure of porcine RBP [38] have been extensively described (see also previous review). The wild type [39^41] and several mutants of human TTR [42^44] and of complexes of the wild type with pharmacologically important compounds [45^47] as well as chicken TTR [48] have been the subject of very detailed X-ray structural studies. The
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Fig. 1. Ribbon representation of the structure of the hexameric complex (RBP)2 TTR determined by X-ray analysis of the chimeric crystals grown using human TTR and chicken RBP. The retinol molecules are represented as CPK models. This view is looking down the z axis of the TTR tetramer as de¢ned by Blake et al. [39]. The x and y axes are in the plane of the ¢gure, the ¢rst is horizontal and the second vertical. They intercept in the center of the TTR channel, the hormone-binding site, which is represented empty in the ¢gure.
two members of the complex are thus structurally very well characterized. TTR, the ¢rst plasma protein whose structure was determined by X-ray di¡raction studies, is a tetramer of four identical subunits, each 127 amino acids long (see Fig. 1). In the tetramer, the four monomers do not occupy equivalent positions but are organized as a dimer of dimers. Each monomer presents two extensive L sheets, each composed of four strands that are all antiparallel with one exception [39^41]. Two monomers form then a very stable dimer by extending their two L sheets through hydrogen bonding that involves the four strands (two from each monomer) at the edges of the two subunits. The two dimers of the tetramer are separated by a channel and in contact through î in symmetry related loops. The channel, about 10 A diameter, has been shown to be the ligand-binding site [39]. The three orthogonal molecular twofold axes of TTR have been designated x, y and z [39], the latter being coincident with the ligand binding channel of the molecule. The RBP molecule is shaped like a calyx made up of eight strands of antiparallel L sheet which are
followed topologically by a short alpha helical segment ([35]; see also previous review). Into this calyx, the retinol molecule binds with the L ionone ring buried deepest and with the alcohol moiety pointing to the outside on the surface of the molecule. Recall that the presence of the vitamin bound to RBP is correlated to the formation of a more stable complex with TTR [33]. Well re¢ned models of RBP liganded to several di¡erent retinoids are also available [49]. Though it was initially thought that removal of the retinol molecule from the calyx would result in major conformational changes, X-ray crystallographic studies of human and bovine RBP [36,37] have shown that the transition from holo- to apoprotein involves only very subtle modi¢cations. The most important is a conformational change on the loop extending from amino acids 34 to 37, in particular, Leu 35 and Phe 36. The space left empty by the removal of the vitamin is ¢lled in both cases by the aromatic ring of Phe 36 and solvent molecules and the movement of the Phe side chain drags the nearby amino acids into positions which are di¡erent from those adopted in the holoprotein.
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3. Crystal structures of the complex Two models of the TTR-RBP complex have been determined from X-ray di¡raction data which extended in both cases to moderate resolution (3.1 î respectively). The two structures deterand 3.2 A mined are that of the chimeric complex humanTTR-chicken-RBP [50] and that of the homologous complex human-TTR-human-RBP [51]. For the chimeric complex it was found that the imposition of a stoichiometry of two RBP molecules per TTR tetramer was essential for successful crystallization [52]. An even higher ratio of 2.5^3 RBP molecules per TTR tetramer was used for the crystallization of the human homologous complex. In both cases the species found in the crystals was the hexamer TTR(RBP)2 but the quaternary structure of the complex in the two cases turned out to be di¡erent. Fig. 1 is a ribbon representation of the chimeric hexamer in which the view is looking down the z axis of the TTR tetramer as de¢ned by Blake et al. [39]. The x and y axes are in the plane of the ¢gure. The x axis is horizontal and separates the two TTR dimers that assembled constitute the tetramer. The y axis is vertical and in the chimeric complex it is the twofold axis that relates the two RBP molecules as should be apparent from the ¢gure. Thus, in this complex the
two RBP molecules are found to interact most extensively with the same TTR dimer. In the homologous human complex, the twofold axis which is preserved is the z axis instead and so the two RBP molecules are found to interact most extensively with the opposite dimers of the TTR tetramer. The reason why two di¡erent twofold axes are preserved in the two crystals studied is not clear but at this point it is probably pertinent to emphasize that the molecular species we have to imagine circulating in plasma is the pentamer and not the hexamer which was created arti¢cially in vitro in both cases and which had to have a higher symmetry to facilitate the packing of the complexes in the crystal lattices. In both the chimeric and the human homologous complex, the structure of the hexamer precludes the possibility of the binding of a third and fourth RBP molecules. In the chimeric hexamer in which the y axis is the preserved twofold, the two RBP molecules bind to the TTR tetramer along an axis which is parallel to the x axis (Fig. 1) and which is su¤ciently close to it to hinder the potential binding of the two other RBP molecules that would be required to satisfy this symmetry element in an octamer. Something analogous happens in the homologous human hexamer in which the two RBP molecules bind on the opposite sides of the x axis but again too close to it
Table 1 Signi¢cant contacts in the TTR-RBP complex in the two structures determined by X-ray crystallography RBP
Distance chimeric complex î) (A
Distance homologous complex î) (A
TTR
E E E E E E E E E E E E E E E
3.41 4.20 3.45 2.73 4.54 4.07 3.09 2.69 3.38 3.37 3.44 2.80 2.70 3.87 3.15
4.24 5.03 4.04 3.57 4.92 3.68 2.71 3.25 3.46 3.30 3.25 3.59 5.47 4.33 3.48
B (cc) D C (cc) A C (cc) A A (cc) C A (cc) C A (cc) C B (cc) D B (cc) D B (cc) D B (cc) D C (cc) A B (cc) D A (cc) C B (cc) D B (cc) D
^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
35 (Leu) [CD2] 67 (Trp) [CZ2] 67 (Trp) [CD2] 89 (Lys) [NZ] 89 (Lys) [NZ] 91 (Trp) [NE1] 95 (Ser) [OG] 96 (Phe) [CO] 96 (Phe) [N] 96 (Phe) [CB] 96 (Phe) [CE1] 97 (Leu) [CO] 99 (Lys) [NZ] 99 (Lys) [N] (Retinol) [OH]
(hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc) (hc)
^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
83 (Gly) [CA] 20 (Val) [CG1] 84 (Ile) [CD1] 99 (Asp) [CO] 99 (Asp) [OD2] 100 (Ser) [CO] 114 (Tyr) [OH] 85 (Ser) [N] 114 (Tyr) [OH] 84 (Ile) [CG2] 21 (Arg) [CG] 85 (Ser) [OG] 99 (Asp) [OD2] 85 (Ser) [OG] 83 (Gly) [CO]
The RBP molecule selected in both cases is chain E and it is in contact with the chains de¢ned A, B, and C in the TTR tetramer of the chimeric complex. In the homologous complex the three equivalent TTR monomers are de¢ned as D, C and A. The ¢rst column gives the distance in the chimeric complex [50] and the second the distance in the homologous human complex [51].
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69
Fig. 2. Schematic representation of the main protein-protein interactions found in the chimeric complex. An asterisk identi¢es Ile 84 which is present in the contact area of the di¡erent TTR monomers, B and C, that are part of the two separate dimers of the tetramer.
to allow for the formation of the octamer. Thus the two structures are consistent with a stoichiometry of a maximum of two RBP molecules per TTR tetramer. The main amino acids involved in the intermolecular contacts between an RBP molecule and the TTR tetramer, initially identi¢ed in the chimeric complex on the basis of a distance cuto¡ [50], are listed in Table 1, along with the distances measured in that model. Table 1 also gives the distances measured between the equivalent atoms in the homologous human complex [51]. From the table one can conclude that the two X-ray structures are substantially in agreement with one another but there is one major signi¢cant di¡erence between the two models. In the homologous human complex, the last ¢ve RBP amino acids, which are not present in chicken RBP (the
species used for the chimeric complex), are found to interact with the TTR tetramer in the case of one of the RBP molecules in the hexamer and not in the other. Thus, in the homologous complex, one of the RBP molecules interacts more extensively with the TTR tetramer than the other so that in fact the molecular twofold axis in the hexamer is not strictly preserved. The physiological signi¢cance of this additional interaction is discussed in [51]; the reasons for this asymmetry in the binding of the two RBP molecules in the case of the human homologous complex are not known. Fig. 2, taken from [50], is a cartoon representation of the most important interactions found in the chimeric complex between an RBP molecule and the TTR tetramer. Notice that the RBP molecule interacts with three di¡erent TTR monomers and that
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there is one amino acid, Ile 84, in two TTR monomers, one from each dimer, that participates in the interaction. In the homologous complex, two other amino acids belonging to the two di¡erent TTR dimers were identi¢ed as participating in the interaction: Val 20 and Arg 21 [51]. The RBP amino acids present in the protein-protein recognition area are located at the entrance of the L barrel and the retinol hydroxyl group has its oxygen at a distance of about î from the polypeptide chain of a TTR mono3^3.5 A mer. A fairly large hydrophobic area is situated between the B and C TTR monomers and the RBP E molecule in the chimeric complex. These hydrophobic contacts are among amino acids which are exposed to the solvent in the uncomplexed proteins and are therefore a very important driving force in complex formation. There are 27 amino acid di¡erences in the primary structure of chicken and human TTR and 23 di¡erences in the sequences of chicken and human RBP [53^56]. Of those amino acids, only one in the case of TTR, amino acid number 114 (a Tyr in human TTR and a Phe in the chicken counterpart) is found in the region of interprotein contacts of the chimeric complex. In the case of RBP it is also only one of the di¡erent amino acids which is found to participate in the intermolecular contacts in the homologous human complex [51]: amino acid number 64 which is a Leu in human RBP and a Phe in the chicken protein. This very high degree of conservation in the regions of protein-protein contacts in the complex emphasizes the physiological relevance of complex formation and its importance in animal physiology. 4. Experimental evidence that supports the X-ray models of the complex The two X-ray models described above are in agreement with the results of a series of experimental studies of which we will give a brief account here. It is known that the a¤nity of RBP for TTR is enhanced at high and reduced at low ionic strength [20] which is consistent with the participation of a hydrophobic area in the intermolecular contacts. Complex formation stabilizes the binding of retinol to RBP [57,58] and substitution of this ligand can
impair or even prevent the interaction of the two macromolecules [33,59] in agreement with the participation of the retinol hydroxyl group in the contacts with TTR. The binding of thyroxine to TTR is identical in the complex and in the isolated protein in accordance with the fact that the ligand binding site is located in an area that does not participate in any way in the macromolecular interactions [20,60]. Chemical modi¢cation of the tryptophan residues of RBP isolated and in the complex has shown that there is at least one Trp which reacts in the isolated protein and is instead protected upon complex formation, which implies that it is located in the area where the protein-protein contacts are established [61]. The identity of this residue was not established chemically but the two X-ray models of the complex discussed above have the same two Trp residues in the contact area: Trps 67 and 91. Acetylation of holo RBP with N-acetylimidazole (using conditions under which both Lys and Tyrs react) disrupts its binding to TTR and deacetylation of the tyrosines with hydroxylamine fails to produce RBP with a normal a¤nity for TTR [62]. This result indicates that there is at least a Lys and that there are no Tyrs present in the RBP area that participates in the protein-protein contacts. As Table 1 shows, the two models have Lys 89 and 99 in the surface in contact with TTR. The reduced a¤nity of apo RBP for TTR can also be explained by the two X-ray models. X-Ray structural studies had shown that in the holo to apo transition in RBP the only signi¢cant change involves amino acids 34^37 [36,37]. This area of the molecule, and in particular Leu 35, is present in the proteinprotein contact area in both X-ray models. Site directed mutagenesis studies on human RBP have con¢rmed the role of Leu 35 and also shown that in the double mutant in which Leu 63 and Leu 64 are changed to Arg and Ser the ability to bind to TTR is reduced [63]. Deletion of loop 92^98 resulted in complete loss of the ability to interact with TTR. Both X-ray models are consistent with the crucial role of this loop in the intermolecular interactions. More than 50 pathological variants of human TTR have been described [2,64]. They are mostly associated to the severe disease called familial amyloidotic polyneuropathy [2,42] but also to senile systemic amyloidosis [65] and euthyroid hyperthyroxine-
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mia [43]. According to the two X-ray models of the complex, in three of these mutants the residues altered [66^68] are involved in the interactions with RBP. In the ¢rst of this mutants, identi¢ed in Japan, the mutation changed Tyr 114 into a Cys [66] and a detailed characterization of the altered protein was not reported. The second mutant, in which Val 20 was substituted by an Ile, was associated with lateonset cardiac amyloidosis [67]. The plasma levels of RBP in this patient were not investigated. The third mutation, found in patients with the disease called Indiana type hereditary amyloidosis, has the substitution Ile 84CSer and the characteristic of showing markedly decreased levels in serum RBP [69]. A quantitative determination of the dissociation constant of the complex of this mutant with normal RBP showed that the a¤nity of the altered TTR for normal RBP is negligible, which explains the reduced RBP concentration found in the plasma of these patients [70]. In both X-ray models of the complex, this residue, Ile 84, is present twice at the interface with RBP and the monomers to which the residues belong are in di¡erent TTR dimers, which explains the dramatic e¡ect that this single mutation has on the stability of the complex. When the interactions of RBP with TTR were analyzed using mass spectrometry, the main conclusions reached were found to be in close agreement with the X-ray model of the chimeric complex [31]. References [1] M. Kanai, A. Raz, D.S. Goodman, J. Clin. Invest. 47 (1968) 2025^2044. [2] Y. Ingenbleek, V. Young, Annu. Rev. Nutr. 14 (1994) 495^ 533. î . Albertsson, B. Hansson, Eur. J. Biochem. 99 [3] G. Fex, P.-A (1979) 353^360. [4] D.S. Goodman, Plasma retinol-binding protein, in: M.B. Sporn, A.B. Roberts, D.S. Goodman (Eds.), The Retinoids, vol. 2, Academic Press, New York, 1984, pp. 41^88. [5] D.R. Soprano, K.J. Soprano, D.S. Goodman, J. Lipid Res. 27 (1986) 166^171. [6] P.W. Dickson, G.J. Howlett, G. Schreiber, J. Biol. Chem. 260 (1985) 8214^8219. ê brink, P.A. [7] H. Ronne, C. Ocklind, K. Wiman, L. Rask, B. O Peterson, J. Cell Biol. 96 (1983) 907^910. [8] M. Navab, J.E. Smith, D.S. Goodman, J. Biol. Chem. 252 (1977) 5107^5114.
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