Structural and mutational analyses of protein-protein interactions between transthyretin and...

Structural and mutational analyses of protein–proteininteractions between transthyretin and retinol-bindingproteinGiuseppe Zanotti1,2, Claudia Folli3, Laura Cendron1,2, Beatrice Alfieri3, Sonia K. Nishida4,Francesca Gliubich1,*, Nicola Pasquato1, Alessandro Negro5 and Rodolfo Berni3

1 Department of Chemical Sciences and Institute of Biomolecular Chemistry-CNR, University of Padua, Italy

2 Venetian Institute of Molecular Medicine, Padua, Italy

3 Department of Biochemistry and Molecular Biology, University of Parma, Italy

4 Department of Medicine, Federal University of Sao Paulo, Brazil

5 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: [email protected]

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: [email protected]

*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 imme-

diate release: PDB 3BSZ for the TTR–RBP–

Fab complex and PDB 3BT0 and 3CXF

for the V20S and Y114H TTR variants,

respectively

(Received 23 July 2008, revised

22 September 2008, accepted 25

September 2008)

doi:10.1111/j.1742-4658.2008.06705.x

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 transthy-

retin, human holo-retinol-binding protein and a murine anti-retinol-binding

protein Fab are assembled according to a 1 : 2 : 2 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 com-

plex formation with retinol-binding protein has been examined by muta-

tional 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 hydro-

phobic interactions and a high degree of complementarity between retinol-

binding 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 reti-

nol-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 retinol-

binding protein are present in piscine transthyretin, consistent with the lack

of interaction between retinol-binding protein and transthyretin in fish.

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 (TTR), a homotetramer of approxi-

mately 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-hydroxyiso-

urate hydrolase, an enzyme distributed in several

prokaryotes and in several eukaryotic lineages and

involved in purine metabolism [2–6]. The extracellu-

lar transport of retinol (vitamin A alcohol) is specifi-

cally 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 reti-

nol–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 mol-

ecule. In turn, the stability of the RBP–TTR com-

plex 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 uncom-

plexed apoRBP molecule, resulting from the delivery

of retinol, being selectively cleared from the circula-

tion by glomerular filtration. TTR has been associ-

ated with human diseases. It is one of several

proteins that can produce the extracellular accumula-

tion 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 differ-

ent 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 com-

plexes have also been determined, both characterized

by a 1 : 2 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 sub-

units. Each monomer is composed of eight anti-par-

allel b-strands (A–H), arranged in a topology similar

to the Greek key b-barrel, with a short a-helixlocated 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 eight-

stranded up-and-down b-barrel. The vitamin mole-

cule 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 con-

nect 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 estab-

lished 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 determi-

nants 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.

Transthyretin–retinol-binding protein interactions G. Zanotti et al.

5842 FEBS Journal 275 (2008) 5841–5854 ª 2008 The Authors Journal compilation ª 2008 FEBS

Results

Structure of a complex between human TTR,

human holoRBP and an anti-RBP Fab

The crystal structure of the human TTR–RBP com-

plex 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 replace-

ment starting from the available high resolution crys-

tal structures of RBP and TTR, is of reasonable

quality. The anti-RBP Fab is bound to an RBP epi-

tope that is well separated from the TTR-binding

determinants present in the RBP molecule. It inter-

acts with RBP on the side opposite to that involved

in the binding to TTR (Fig. 1A), so that the interac-

tions 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 trans-

verses the TTR molecule. RBP and TTR substan-

tially maintain the structure they have in the

uncomplexed state, with only minor changes in con-

tact regions.

The region of entrance of retinol into the b-barrelcavity 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 simul-

taneously with three TTR subunits (Table 1 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 com-

plex with the hydrophobic patch formed by the resi-

dues 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 sepa-

rated RBP and TTR molecules, respectively.

The Fab–RBP interaction involves the region pre-

ceding 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 2. 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 inter-

acting 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 dif-

ferent 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. 2. The func-

tional and structural consequences of several TTR

point mutations on the RBP–TTR interactions have

been investigated. The values of the dissociation con-

stants for several complexes between human TTR vari-

ants and human RBP, as determined by means of

fluorescence anisotropy titrations (Fig. 3A), are

reported in Table 3. For some TTR mutations that

abolish or weaken significantly the RBP–TTR interac-

tions, 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 sub-

units (B and C, according to our designation of TTR

subunits) are located at the center of a large hydropho-

bic patch in the contact area between TTR and RBP,

so that their replacement by a hydrophilic residue is

expected to significantly impair protein–protein inter-

actions. Accordingly, the V20S mutation has been

found to almost abolish the binding affinity between

RBP and TTR (Table 3 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,

G. Zanotti et al. Transthyretin–retinol-binding protein interactions


A

B

C

Fig. 1. 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 approxi-

mately 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 clock-

wise, respectively, compared to the previous view. Residues 163–169 and the region preceding the a-helix contribute to the formation of

the RBP epitope.



significant differences are observed for loop region

98–103, which connects b-strands G to F and is flexi-

ble 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 Caatom 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 estab-

lished with RBP.

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 3 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 posi-

tion in a hydrophobic patch of TTR involved in hydro-

phobic interactions with RBP; its replacement by a

serine can perturb the polarity of the microenvironment

at the interface, thereby impairing protein–protein rec-

ognition. 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 com-

prising residue 84, with the disruption of the a-helixitself [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 inter-

action 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

Table 1. 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 TTR chain B TTR chain C

W91 S100

V93 R103a

S95 V122

K99 D99a, S100a

L35 L82, G83

L63 L82, G83, I84

L64 L82a, R21

S95 Y114b

F96 I84, S85b, Y114a R21

L97 S85a

Q98 S85a

K99 S85b

W67 G83, I84

Retinol 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.

Table 2. 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 Fab chain L Fab chain H

R 163 F36, S95a, Y100a

Q164 N34a, R54b

Y165 R54b Y103

R166 Y53, R54a, N57a

Q149 F101, Y52, T30b, S31b

K150 D55b

Q156 D102a

R163 D102b,Y103a

L167 Y103b

a 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.



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

to an approximately five-fold decrease in binding affin-

ity caused by the mutation (Table 3). It can be specu-

lated that the loss of interactions caused by the

A

B

Fig. 2. 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 acces-

sion 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].



mutation may be partially compensated by a novel

hydrophobic interaction in a hydrophobic patch

involving Ala85.

D99A and S100E TTR variants

These are important mutations because each of them

causes an approximately 20-fold decrease in binding

affinity of TTR for RBP (Table 3 and Fig. 3A). Both

replaced residues interact with Lys99 of RBP and,

moreover, Ser100 is close to Trp91 (Table 1). Conse-

quently, 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 involv-

ing 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.

Y114F and Y114H TTR variants

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 3

and Fig. 3A). We have also determined the crystal

structure of the amyloidogenic Y114H variant: no sig-

nificant 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 orienta-

tion 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 conforma-

tional rearrangements that result in stronger hydro-

A

B

Fig. 3. 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 dissoci-

ation 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) Ste-

reo view showing the superposition of the Ca chain traces of wild-

type 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.

Table 3. 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].b 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.



phobic interactions, possibly explaining the higher

affinity between RBP and this TTR variant. The

replacement of Tyr114 by a potentially charged histi-

dine could have a marked effect on the surface poten-

tial; 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 sig-

nificant conformational changes compared to the un-

complexed 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 correspond-

ing human proteins present in the complex. The struc-

ture 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 Swiss-

Model 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 muta-

tions affecting interacting residues (Table 3 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 3 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 differ-

ences 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 confor-

mational 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 rela-

tively 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. 4. 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.



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 weaken-

ing 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 hydro-

philic for hydrophobic side chains in TTR contact

regions generally have a rather pronounced dissociat-

ing effect on the RBP–TTR complex, consistent with

the important role played by interfacial apolar inter-

actions. In general, the changes in the TTR molecule

induced by amyloidogenic mutations do not interfere

with the interactions between RBP and TTR, unless

the mutations are located in contact areas. The amyloi-

dogenic 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 do not

cause large conformational changes in the TTR mole-

cule that might affect indirectly the RBP–TTR recogni-

tion. Conversely, the amyloidogenic I84S mutation,

which affects a residue that is crucial for protein–pro-

tein interactions, causes the lack of recognition

between RBP and TTR and an altered plasma trans-

port 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 homo-

tetramer with virtually four identical binding sites for

RBP, a 1 : 1 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 tetra-

mer. The binding of two RBP molecules to an uncom-

plexed 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 macro-

molecular organizations, both accounting for the 1 : 2

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

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 assem-

bly 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 observa-

tion, 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 res-

idues 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 inter-

action 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



acid replacements at positions 82, 84, 99 and 100 in

piscine TTR compared to the human and chicken pro-

teins are drastic and are present at positions critical

for the interaction between RBP and TTR (Table 1

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 3 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 appear-

ance in terrestrial vertebrates of the TTR function of

cotransport of retinol through the interaction with

holoRBP in plasma, in addition to that of the distribu-

tion 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 pre-

pared 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 reac-

tion was treated with DpnI (New England Biolabs, Beverly,

MA, USA) to digest the parental DNA template. This pro-

cedure allowed us to select the newly synthesized and

potentially mutated plasmids. The products of each diges-

tion were used to transform Escherichia coli XL1 Blue cells.

Single clones were then sequenced to confirm the occur-

rence of the desired mutation. Finally, mutant plasmids

were electroporated into E. coli BL21 (DE3) cells. The

expression of TTR variants was induced by 1 mm isopropyl

thio-b-d-galactoside and, after incubation for 4 h at 30 �C,cells were disrupted by sonication. TTR variants were puri-

fied as described for wild-type TTR [39].

Determination of the amino acid sequences of

anti-RBP Fab variable domains

Total RNA obtained from the cell line producing the anti-

RBP murine monoclonal antibody A8P3 [47] was subjected

to retrotranscription into cDNA employing the Superscript

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 polariza-

tion 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 concen-

trated 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 concen-

tration of TTR, and Amax and Ao are the two limiting

anisotropy values (i.e. in the presence of an excess saturat-

ing TTR and in the absence of TTR, respectively). Binding

data were analyzed as described [12]. Fluorescence anisot-

ropy measurements were carried out with a LS-50B spectro-

fluorometer (Perkin-Elmer, Waltham, MA, USA).

Crystallization, data collection, structure

determination and refinement for the

TTR–RBP–Fab complex and the V20S and

Y114 TTR variants

Crystallization and preliminary X-ray data for the macro-

molecular complex formed by human transthyretin, human

holoRBP and a murine anti-RBP Fab have been reported



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 noncrystallo-

graphic 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, respec-

tively, 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, account-

ing for a total of 10 polypeptide chains (one TTR tetramer,

two RBP and two Fab) and a molecular mass of approxi-

mately 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–

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 2 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 stereo-

chemistry, 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 4 and 5. 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 com-

plex, 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 dis-

cussions. 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, Italy.

Table 4. Data collection. Values in parentheses are for the outer resolution shell.

TTR–RBP–Fab complex V20S TTR variant Y114H TTR variant

Space group and

cell parameters

C222, a = 159.62,

b = 223.18, c = 121.43

P21212, a = 42.00

b = 83.81, c = 65.58

P21212, a = 42.56

b = 86.28, c = 64.83

Resolution (A) 15.72–3.36 (3.51–3.36) 65.5–1.59 (1.63–1.59) 64.8–2.30 (2.42–2.30)

Independent reflections 25746 (1622) 30276 (2071) 9936 (1599)

Multiplicity 3.0 (2.6) 3.2 (2.0) 5.9 (6.2)

Completeness (%) 84.4 (77.7) 99.1 (92.9) 99.3 (100)

<I ⁄ r(I )> 3.3 (1.9) 17.3 (2.1) 5.2 (3.9)

Rmergea 0.16 (0.34) 0.051 (0.423) 0.095 (0.156)

a Rmerge ¼ Rhkl Ihkl� Ihklh ij jRIhkl

.

Table 5. Refinement statistics. Values in parentheses are for the

outer resolution shell.

TTR–RBP–Fab

complex

V20S TTR

variant

Y114H TTR

variant

Protein atoms 13 058 1765 1783

Solvent molecules ⁄ligand atoms

334 ⁄ 42 165 ⁄ 0 94

Rcryst.a 0.239 (0.313) 0.209 (0.277) 0.218 (0.258)

Rfree 0.312 (0.357) 0.229 (0.303) 0.281 (0.326)

Mean B value (A2) 37.5 17.8 23.3

Ligands mean B

value (A2)

13.4 – –

rmsd from ideal

values

Bond lengths (A) 0.010 0.011 0.006

Bond angles (�) 1.5 1.29 1.3

a Rcryst ¼ Rhkl Foj j�k Fcj jj jRhkl Foj j , where |Fo| and |Fc| are the observed and calcu-

lated structure factor amplitudes for reflection hkl, applied to the

work (Rcryst) and test (Rfree) (7% omitted from refinement) sets,

respectively.



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Supporting information

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 miss-

ing material) should be directed to the corresponding

author for the article.



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