the transthyretin-related protein: structural investigation of a novel protein family

Journal of Structural Biology 155 (2006) 445–457

www.elsevier.com/locate/yjsbi

The transthyretin-related protein: Structural investigation of a novel protein family

Erik Lundberg, Stefan Bäckström 1, Uwe H. Sauer, A. Elisabeth Sauer-Eriksson ¤

Umeå Centre for Molecular Pathogenesis, Umeå University, SE-90187 Umeå, Sweden

Received 5 January 2006; received in revised form 3 April 2006; accepted 4 April 2006Available online 8 May 2006

Abstract

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric trans-port protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved inpurine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 Åresolution in the presence of zinc, and the other at 2.1 Å resolution in the presence of zinc and bromide. The structures revealed Wve zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chainsof residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98,and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 alsobinds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-termi-nal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues formdirect hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-bind-ing channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.© 2006 Elsevier Inc. All rights reserved.

Keywords: Transthyretin-related protein; TRP; Escherichia coli; Crystal structure; Transthyretin

1. Introduction

The transthyretin-related proteins (TRP)2 constitute afamily of proteins structurally related to transthyretin(TTR) and are found in a wide variety of species. Organ-isms harboring TRP include bacteria, plants, fungi, andanimals (including vertebrates) (Eneqvist et al., 2003). Incontrast, TTR seems to be vertebrate speciWc (Power et al.,2000). The C-terminal four amino acids Y-[RK]-G-[ST]constitute a signature sequence for the TRPs which distin-

* Corresponding author. Fax: +46 90 778007.E-mail address: [email protected] (A.E. Sauer-Eriksson).

1 Present address: Department of Medical Biochemistry and Biophysics,Umeå University, SE-90187 Umeå, Sweden.

2 Abbreviations used: TRP, transthyretin-related protein; hTTR, humantransthyretin; EcTRP, Escherichia coli TRP; HIUH, hydroxyisouratehydrolase; MR, molecular replacement; r.m.s., root mean square.

1047-8477/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.jsb.2006.04.002

guishes the TRPs unambiguously from TTR and other pro-teins listed as transthyretin-like in databases (Eneqvistet al., 2003).

To date 91 TRP sequences from 86 diVerent species havebeen identiWed. Most are of bacterial origin, but some newvertebrate species have been identiWed as well, such as TRPfrom Gallus gallus (chicken, XP_414218). Furthermore,TRP is found at the C-terminal end of a 751 amino acidprotein from Canis familiaris (dog, residues 631–751,XP_546767). The sequence of the N-terminal 630 aminoacids is similar to cadherin-1. The canine and mouse TRPproteins (XP_133915, AAH51545) have approximately80% of the amino acids in common. Interestingly, the TRPsequence, with 78% sequence identity to dog and mouseTRP, is found in the central part of a 346 amino-acid pro-tein from Pan troglodytes (chimpanzee, XP_511183). How-ever, the last eleven C-terminal residues, corresponding tothe entire �-strand H, are missing in the chimpanzee

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446 E. Lundberg et al. / Journal of Structural Biology 155 (2006) 445–457

sequence. The chimpanzees carry the gene for this proteinon chromosome 16. A similar DNA sequence is alsopresent on the human chromosome 16 (AC133542, nt28134–32298), however, it has been rearranged and seemsto contain only fragments of the TRP sequence (Eneqvistet al., 2003). In insects, we had previously identiWed TRP inDrosophila melanogaster (Eneqvist et al., 2003) and havenow found it also in Drosophila pseudoobscura (fruitXy,EAL25949), Apis mellifera (honeybee, XP_393041), andAnopheles gambiae (malaria mosquito, EAA06561). InD. melanogaster and D. pseudoobscura, TRP ends with anYRGT sequence.

Little is known about the function of TRP. Studies per-formed in Bacillus subtilis showed that the TRP gene pucMis essential for urate oxidase (uricase) activity (Schultzet al., 2001). The gene pucM is co-regulated with pucJ,pucK, and pucL, which are genes encoding proteinsrequired for uric acid uptake and oxidation. In addition,recent studies have identiWed a role of TRP in facilitatinghydrolysis of 5-hydroxyisourate (HIU) to (S)-allantoin(Lee et al., 2005; Ramazzina et al., 2006). This suggests thatTRP actually functions as a hydroxyisourate hydrolase(HIUH) (Lee et al., 2005; Ramazzina et al., 2006), necessaryfor the purine catabolic pathway (Raychaudhuri andTipton, 2002; Raychaudhuri and Tipton, 2003). A sub-fam-ily of TRP is found in Arabidopsis thaliana as well as inmany other plants. These proteins are called transthyretin-like proteins (TTL), and are characterized by their extendedN-terminal region of about 200 amino acids. TTLs are sug-gested to be negative regulators of the transmembranebrassinosteroid receptor in plants. However, this functionis associated with the N-terminal region of these proteins(Li, 2005; Nam and Li, 2004).

The biological function of TTR is to transport and dis-tribute the two thyroid hormones 3,5,3�-triiodo-L-thyronine(T3) and 3,5,3�,5�-tetraiodo-L-thyronine (thyroxine, T4), aswell as vitamin A in complex with retinol-binding protein(Hamilton and Benson, 2001; Palha, 2002). Under certainconditions, TTR can cause disease by misfolding andassembling into Wbrillar aggregates known as amyloids(Costa et al., 1978; Kelly, 1998; Saraiva et al., 1983).Amino-acid point mutations in TTR give rise to familialamyloidotic polyneuropathy (FAP), an autosomal domi-nant amyloid disorder (for reviews see (Benson andUemichi, 1996; Saraiva, 1995)). The three-dimensionalstructure of TTR is well characterized. The protein is ahomotetramer with a central hydrophobic channel in whichthe two thyroxine-binding sites are situated (Blake et al.,1978; Hamilton et al., 1993; Hornberg et al., 2000; Wojtc-zak et al., 1996). Each monomer has the fold of an immuno-globulin-G like �-barrel formed by two �-sheets(comprising �-strands D, A, G, and H and C, B, E, and F,respectively). Dimerization occurs predominantly throughintermolecular main-chain hydrogen bonds between the H�-strand of each monomer, thereby forming a continuouseight-stranded �-sheet. The tetramer forms through hydro-phobic interactions between two dimers, thereby creating a

hydrophobic channel with two identical thyroid hormone-binding sites.

We have determined the three-dimensional X-ray crystalstructure of the E. coli TRP (EcTRP), also called YedX,and compared it to TTR. The overall structure of TRP issimilar to TTR, with structural diVerences found in surface-exposed loop areas and in �-strand D. The dimer–dimerinterface is formed by highly conserved amino acids uniqueto the TRP family, and we believe, in analogy to TTR,that this site comprises the putative substrate/ligand-bind-ing site of TRP. The central channel is shallow andpositively charged in TRP, and constitutes a uniquemetal-binding site.

2. Materials and methods

2.1. Protein puriWcation

Mature, wild-type TRP (i.e., excluding the signal pep-tide) was cloned, expressed and puriWed from E. coli as pre-viously described (Eneqvist et al., 2003). BrieXy, the proteinwas batch puriWed using SP Sepharose (GE Healthcare).Peak fractions were pooled and dialyzed against 50 mMTris–HCl, pH 7.5, and 200 mM NaCl. Protein purity wasassessed by SDS–PAGE to be greater than 95%. The pro-tein was concentrated (Centriprep, Amicon) to »5.0 mg/ml,frozen in liquid nitrogen, and stored at ¡80 °C.

2.2. Metal content analysis

Metal content analysis was performed by inductively-coupled, plasma-sector Weld mass spectrometry for theelements: Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg,Mn, Na, Ni, Pb, S, and Zn (Analytica AB) in buVer con-taining 200 mM NaCl. The analysis was performed on twoseparate protein samples puriWed from bacterial cellsgrown in media with or without the addition of 10 �MZnCl2.

2.3. Crystallization, data collection, and data processing

DiVracting quality crystals of TRP were grown at 18 °Cusing the hanging-drop vapor-diVusion method. The totaldrop volume amounted to 5 �L including: 2�L of the reser-voir solution containing 25% polyethylene glycol 550monomethyl ether (PEG550 MME), 100 mM MES, pH 6.5,and 10 mM ZnSO4, 1 �L of 0.1 M Trimethylamine–HCl(Hampton Research Inc.), and 2 �L of protein solution at5.0 mg/ml. Bromide derivatives were obtained by soakingcrystals in crystallization buVer enriched with 100 mMNaBr for 12 h before data collection.

DiVraction data from zinc and zinc/bromide crystalswere collected at a Wxed wavelength of 0.8033 Å on a165 mm MAR Research CCD detector at beam line X13(EMBL-outstation at DESY, Hamburg, Germany). Thecrystals were supported at 100 K in cryo loops (Sauer andCeska, 1997). Complete X-ray diVraction data sets

E. Lundberg et al. / Journal of Structural Biology 155 (2006) 445–457 447

extending to 1.65 and 2.10 Å resolutions were collected,respectively. DiVraction patterns could be indexed inspace group P21. The asymmetric unit contains one TRPtetramer and approximately 40% (v/v) solvent. The zincdata set was processed and scaled with DENZO andSCALEPACK (Otwinowski and Minor, 1997). The zinc/bromide data set was processed and scaled with XDS andXSCALE (Kabsch, 1993). The data collection statisticsare given in Table 1.

2.4. Structure determination and reWnement

The structure of zinc-bound EcTRP was determined bymolecular replacement methods from data between 17.0and 1.65 Å resolution. A truncated poly-alanine model of

human TTR (hTTR) (PDB Accession Code 1F41,(Hornberg et al., 2000)) was used as search model in CNS(Brünger et al., 1998). Initial reWnement was carried outwith CNS using conjugate gradient minimization and sim-ulated annealing, alternated with manual rebuilding of themodel in O (Jones et al., 1991). ReWnement was performedwith 95% of the data, whereas 5% were used for cross-vali-dation (Brünger, 1992). The Wnal reWnement was carriedout with the program REFMAC, part of the CCP4 pro-gram package (Collaborative Computational Project,1994), using the maximum likelihood residual, anisotropicscaling, bulk-solvent correction, and atomic displacementparameter reWnement (Murshudov et al., 1997), as well asthe translation, libration, screw-rotation (TLS) method(Schomaker and Trueblood, 1968; Winn et al., 2001).

Table 1Statistics of data collection, reWnement, and structural statistics for EcTRP

a Rmerge D �h�i� Ihi ¡ (Ih)�/�h �i(Ih), where (Ih) is the mean intensity of the i observations of reXection h.b R-factor D �hkl�Fobs(hkl)�¡ k�Fcalc(hkl)�/�hkl�Fobs(hkl)�.c From Engh and Huber (1991) (Engh and Huber, 1991).d A bound zink ion enforces the observed geometry of D-Asp31.e The occupancies were reWned with CNS (Brünger et al., 1998).f � is the root mean square density throughout the unit cell.

Zinc derivative Zinc-bromide derivative

(a) Data collectionSpace group P21 P21

Unit cell distances a, b, c (Å) 44.97, 92.67, 58.05 44.72, 92.03, 57.49Unit cell angles (�, �, �) (°) 90.00, 103.86, 90.00 90.00, 103.50, 90.00Resolution range (Å) 17-1.65 (1.73-1.65) 20-2.10 (2.30-2-10)Number of observations 1.012.332 198.239

Friedel unmerged Friedel merged

Number of unique reXections 44.377 49.893 (11.457) 25.512 (5.907)Completeness (%) 80.0 (90.3) 95.8 (91.9) 96.3 (93.6)Rmerge (%)a 5.2 (60.4) 4.1 (18.5) 4.7 (20.0)Mean I/�I 22.6 (2.4) 21.6 (7.3) 27.1 (10.0)

(b) ReWnementResolution (Å) 17.0–1.65 (1.69–1.65) 17.0–2.10 (2.16–2.10)ReXections in the working set 41 300 (3.490) 24 310 (1750)ReXections in the test set 2.197 (189) 1300 (91)R-factor working set (%)b 19.8 (25.7) 17.5 (20.8)R-free test set (%) 23.4 (27.4) 22.9 (28.4)Mean B-factor (Å2) 15.5 19.4r.m.s. deviations from ideal valuesc:

Bonds (Å) 0.009 0.012Angles (deg.) 1.226 1.334

Ramachandran plot:Most favored region (%) 92.3 91.0Additionally allowed region (%) 7.4 8.7Generously allowed region (%) 0.3d 0.3d

Disallowed region (%) 0 0

(c) StructureOccupanciese (%) and {�} valuesf of: Zinc ions (in monomerA,B,C,D) at

Site I (His9-His98) 100{13}, 100{17}, 80{8}, 100{12} 80{7}, 80{7}, 55{4}, 100{9}Site II (His96-His98-Ser114) 30{3}, 35{3}, 100{15}, 75{8} 55{4}, 60{4}, 100{12}, 70{5}Site III (His96-Ser114) 35{3}, 30{3}, 0, 0 45{3}, 40{3}, 0, 30{3}Site IV (Asp61-His89) 55{5}, 100{17}, 35{5}, 100{20} 55{4}, 100{16}, 70{5}, 100{16}Site V (Glu83-Glu87) 50{5}, 60{7}, 50{5}, 70{7} 0, 0, 0, 0

Bromide ions (in monomer A,B,C,D) at site 1 (His9-His98) 95{9}, 85{9}, 65{6}, 100{11}Number of water molecules 271 178Number of sulfate molecules 2 5


Throughout the reWnement, the monomers were treatedindependently and all modeled residues apart from His96were reWned with single conformations and occupanciesset to 1. His 96 was reWned in two conformations as willbe discussed. The structure of the zinc and bromide dou-ble derivative was determined by the diVerence Fouriermethod, and reWned using a similar protocol as describedfor the zinc-bound EcTRP structure. Details of thereWnement statistics for the two structures are shown inTable 1.

Structural superpositions were calculated with lsq-explicit in O (Jones et al., 1991). Dimer–dimer interfaceareas were calculated using the program AREAIMOL(Collaborative Computational Project, 1994). Sequencesfor Fig. 1 were aligned using ClustalW (Higgins et al.,1996). Figs. 2A, B, and 3 were made with the programMOLSCRIPT (Kraulis, 1991). Figs. 4–8 were generatedusing the program PyMol (DeLano, 2002).

2.5. Protein data bank accession number

The structures and structure factors have been depositedin the protein data bank (Accession Codes 2G2N (EcTRP-Zn) and 2G2P (EcTRP-Zn-Br)).

3. Results and discussion

3.1. General description of the TRP structure

The mature EcTRP monomer comprises 114 aminoacids and shares 30% sequence identity with human TTR(hTTR) (Fig. 1). Except for one amino acid deletion in theBC-loop and one in the FG-loop of EcTRP, the diVerentlengths of the proteins are due to extra amino acids at theN- and C-terminal ends of TTR. The structure of EcTRPwas determined by molecular replacement. The quality ofthe initial electron density map was excellent and allowedmore than 90% of the side chains of the EcTRP structure tobe modeled. Inspection of the Ramachandran plot of thereWned coordinates showed that Asp31 in monomer D fallswithin the generously allowed regions (Ramachandran andSasisekharan, 1968). This residue is entirely covered by elec-tron density and the unusual conformation is probably dueto the interaction with a zinc ion from a symmetry-relatedmonomer. The asymmetric unit of EcTRP harbors one tet-ramer comprised of monomers A, B, C, and D. The WnalreWned model includes residues A4-A114, B4-B114, C3-C114, D3-D114, and in addition, 271 water molecules, twosulfate ions, and 18 zinc atoms (Fig. 2).

Fig. 1. Sequence alignment of hTTR and EcTRP. Residues displaying 95% or more sequence identity within the TRP family including 91 sequences from86 species are shown in dark green, while those with more than 95% similarity are light green. Similarly, positions displaying above 95% identity or morethan 95% similarity in the TTR family, including sequences from 24 species, are shown in dark and light blue, respectively. The following groups of aminoacids were deWned as similar: FYW, IVLM, RK, DE, GA, TS, and NQ. Signal peptides were not included in the alignment. The sequence identity betweenhTTR and EcTRP is 30%. Numbering and secondary structure elements are shown for both hTTR and EcTRP. �-Strands are shown as green (hTTR) andblue (EcTRP) arrows, and the �-helix is presented as a red rectangle. The secondary structure deWnitions are based on DSSP (Kabsch and Sander, 1983).The H-strand in hTTR is longer than the H-strand in EcTRP. This is due to direct main-chain interactions in hTTR between residues Val121-Thr123 in�-strand H and residues Arg104-Thr106 in �-strand G. The number of hydrogen bonds which stabilizes the monomer-monomer interface in EcTRP andin hTTR is however identical. Residues with their side-chain surfaces exposed in the putative ligand-binding site of EcTRP are marked with green stars.Sequences were managed with the Biology Workbench (http://workbench.sdsc.edu). (For interpretation of the references to color in this Wgure legend, thereader is referred to the web version of this paper.)

http://workbench.sdsc.edu

http://workbench.sdsc.edu


The EcTRP monomer fold comprises eight anti-paral-lel �-strands, named A–H following the nomenclature ofthe hTTR structure (Blake et al., 1978). The �-strands areorganised into two four-stranded �-sheets with a topologysimilar to the classical Greek key �-barrel. EcTRP alsocontains the short �-helix present in hTTR (Fig. 2). The

Fig. 2. The structure of EcTRP. (A) The structure of the EcTRP dimerbuilt from monomers A and B. Each monomer is composed of one short�-helix and eight anti-parallel �-strands organized in a �-sandwich. Twomonomers dimerize through interactions involving the H-strands and theF-strands. (B) The structure of the EcTRP tetramer. Two extended 8-stranded �-sheets (D-A-G-H-H-G-A-D), one from each dimer, interactface-to-face, which create the putative ligand-binding sites of EcTRP(indicated with red arrows). The 18 zinc ions are shown as grey spheres.Side-chains for the residues interacting with the zinc ions are shown assticks. Sulfate ions bound to the TRP structure are shown as red andgreen balls-and-sticks.

four monomers in EcTRP are very similar as can bededuced from the low root mean square (r.m.s.) diVerencesranging from 0.43 Å (comparing monomers A and B) to0.51 Å (comparing monomers A and C), when superim-posing the main chain N, C�, C, and O atoms of residuesAsn4-Ser114. Two TRP monomers dimerize throughdirect main-chain hydrogen bonds between residuesTyr106-Arg112 situated in the H-strands (correspondingto residues Tyr114-Ala120 in hTTR), and side-chain inter-actions between residues Glu83–Glu87 situated in theF-strands (residues His90-Val94 in hTTR) (Fig. 2A). Sec-ondary structure assignment suggests that the H-strand inEcTRP is slightly shorter than the H-strand in hTTRalthough the number of hydrogen bonds that stabilize themonomer-monomer interface is identical. The extended

Fig. 3. Superposition of C� atoms of EcTRP and previously determinedstructures of TTR. The orientation of the monomers is similar to the onefor the right monomer in Fig. 2A. Most of the structural similaritiesbetween EcTRP (red-brown) and hTTR (dark blue) lie within the A, B, Gand H-strands, while most of the diVerences lie in the surface exposedloops. The main chain N, C�, C, and O atoms of EcTRP deviate fromthose of hTTR, Wsh TTR (cyan), rat TTR (yellow), and chicken TTR(dark green) with 0.6, 0.7, 0.7, and 0.9 Å, respectively, when superimposedon TRP residues 5–12, 17–29, 34–48, 61–88, and 97–111. Due to the dis-torted �-helical structure in chicken TTR, section 61–88 was furtherdivided in sections 61–68 and 83–88 for the superpositioning. The largeststructural deviation between EcTRP and TTR is situated at �-strand Dand the following DE-loop deWned by residues Arg47 and Tyr62.


�-strand conformation of the H-strand in hTTR is due tomain-chain contacts, made between residues Val121-Thr123 in �-strand H and Arg104-Thr106 in �-strand G.The tetrameric form of EcTRP is formed mainly byhydrophobic contacts involving residues situated at theAB- and GH-loops (Fig. 2B). The interactions result inthe formation of a channel, which in TTR comprises thethyroid hormone-binding sites. Each of the 271 watermolecules in EcTRP makes at least one hydrogen bondwith the protein within proper hydrogen-bonding dis-tances of less than 3.5 Å and has temperature factorsbelow 60 Å2.

3.2. Comparison of TRP with TTR

Crystal structures of TTR are available from four spe-cies: human (Blake et al., 1978; Hamilton et al., 1993;Hornberg et al., 2000), chicken (Sunde et al., 1996), rat(Wojtczak, 1997), and sea bream (Eneqvist et al., 2004;Folli et al., 2003). The overall structure of the EcTRPmonomer is very similar to the monomers of the fourTTR proteins (Fig. 3). All structural diVerences betweenEcTRP and hTTR are located in the BC-loop, CD-loop,�-strand D, the DE-loop, and in the FG-loop, i.e., at sur-face exposed loops of the tetramer not involved in tetra-mer association. The AB-loop, EF-loop (including theshort �-helix), and the GH-loop are structurally almostidentical. These loop regions point towards the dimer–dimer interface, and are important for tetramer stabilityand ligand binding.

In the human TTR structure, water molecules W1–W36play a signiWcant role in stabilizing the monomer (W1–W12), the dimer interface between monomers A and B(W13–W20), and the dimer–dimer interface between dimersAB and CD (W21–W36) (Hornberg et al., 2000). Of thesewater molecules, only those that directly bridge the F andF� strands in the protein dimer interface are observed in allstructures. For EcTRP, we identiWed a total of 26 watermolecules as being important for the stability of the mono-meric (W1–W4, one buried water molecule per monomer),dimeric (W5–W10, three per dimer) and tetrameric (W11–W26) forms. With only 16 waters (W11–W26), EcTRP hassigniWcantly fewer positioned in its hydrophobic channelcompared to hTTR, which has 32 waters (hTTR has adimer and not a tetramer in the asymmetric unit therefore16 symmetry-related water molecules generated from W21–W36 are also situated in the channel of the tetramer). InhTTR, there are four water molecules buried within thecore of each monomer, forming hydrogen bonds to the bur-ied side chains of His88 and Thr75. In EcTRP, Phe81 occu-pies the position of His88, and only one buried watermolecule is observed in EcTRP.

In hTTR, the area including the CD-loop and the D-strand (or the “edge-strand” area) is of particular interestsince structural changes initiated at this site seem to be aprerequisite for amyloid-Wbril formation (Eneqvist et al.,2000; Olofsson et al., 2003; Sebastião et al., 1998; Seraget al., 2001; Serpell et al., 1996). This is the only area whereEcTRP signiWcantly diVers from the structure of hTTR.This dissimilarity is most apparent in �-strand D and the

Fig. 4. The conformation of the �-strand D in EcTRP and hTTR. The most signiWcant structural diVerences between EcTRP and hTTR are found in theD-strand area. (A) In TRP, four hydrogen bonds are formed between main-chain atoms of the A- and D-strand, and two are formed between the sidechain of Asn4 and the main chain of Trp52. (B) In hTTR only three hydrogen bonds involving main-chain atoms are present in the corresponding area. Inaddition, hTTR has two water molecules bridging main-chain atoms situated in �-strands A and D.


DE-loop areas comprising residues Arg47-Tyr62 (Fig. 3).The interactions between the B- and C-strands in EcTRPare similar to the ones present in hTTR, and diVerences arepredominantly found in the interaction area between�-strand A and D. In the hTTR monomer, two watermolecules bridge the A- and D-strand through indirecthydrogen bonds (Fig. 4). These water molecules are notobserved in the EcTRP structure, where the A- and D-strand are closer. Four direct hydrogen bonds are formedbetween the main-chain atoms of the two strands inEcTRP. Three of these are equivalent to bonds present inhTTR, and one is a bond formed directly between themain-chain nitrogen of Leu51 to the carbonyl oxygen ofLeu6 that is not observed in hTTR. This arrangementleads to a structurally more ordered �-strand D in EcTRP,which seems possible due to the substitution of Gly57 inhTTR to Ala50 in EcTRP.

The N-terminus of EcTRP is six amino acids shorterthan the human TTR (Fig. 1). In both cases the N-terminalresidues are Xexible, as indicated by the lack of electrondensity. The Wrst well-deWned residue in hTTR is Cys10(Hornberg et al., 2000). Its sulfur atom forms a hydrogenbond to the main-chain nitrogen atom of Gly57. This bondis weak, and various post-translational modiWcations ofCys10 have been observed in ex vivo hTTR protein variantscausing amyloidosis (Ando et al., 1996; Kishikawa et al.,1996; Suhr et al., 1998; Suhr et al., 1999). In vitro studiesalso show that cysteine modiWcations have implications forthe stability of the protein, i.e., they increase the propensityfor protein misfolding and aggregation (Altland et al., 2004;Altland et al., 1999; Zhang and Kelly, 2003; Zhang andKelly, 2005). Histochemical investigations of transgenicmice show that Cys10 plays a crucial role in hTTR amyloi-dogenesis in vivo (Takaoka et al., 2004). Our analysis of theN-terminal region on EcTRP shows that two hydrogenbonds are formed from the side chain of Asn4 (correspond-ing to Cys10 in TTR) to the main-chain carbonyl oxygen ofand nitrogen of Trp52. Thus it is likely that the side chainof Asn4 is strongly coupled to the D strand. However, inthe C and D monomers of the tetramer, crystal packinginteractions between residues C-Gln3 and D-Gln3 shift theposition of C-Asn4 and D-Asn4 by 2.4 and 1.7 Å, respec-tively. One should note that the lack of two hydrogenbonds from Asn4 to the main chain of �-strand D does notlead to a structural shift of the latter �-strand.

The surface below the two BC-loops in each dimer hasa topology referred to as the saddle (Hornberg et al.,2000). The concave side of the saddle is formed byexposed residues that are part of the two interactingF-strands (Fig. 2). So far, no function could be assignedto the saddle. However, residues Lys35 and His90 in bothmonomers of hTTR, are able to interact with chlorideand iodide ions (Hornberg et al., 2005). While the Lys35residue is conserved in EcTRP, the position of His90 isreplaced by Glu83. The saddle of EcTRP binds one sul-fate per dimer with the ion positioned right at the two-fold axis. The sulfate ion thus stabilizes the dimer by

interacting with the side-chains of Trp34 and Arg63 fromboth monomers (Fig. 5).

3.3. Putative ligand-binding sites in TRP

TTR functions as a transport protein for thyroxineand other thyroid hormones. Two thyroid hormone-binding sites are situated at the hydrophobic channelbetween monomers A, C and B, D, respectively (Wojtc-zak et al., 1996). Based on sequence similarities (seeFig. 1) we postulated that TRP also functions as a trans-port protein with its ligand-binding site in the channel(Eneqvist et al., 2003). In support of this hypothesis,amino acids that are conserved within the TRP family,but substituted compared to corresponding residues inTTR, are all part of the putative ligand-binding site(Eneqvist et al., 2003).

In hTTR, thyroxine interacts with seven conserved resi-dues from two symmetry-related monomers at the tetramerinterface (Wojtczak et al., 1996). Thyroxine makes hydro-phobic interactions with Leu17, Ala108, Ala109, andLeu110, and polar contacts to the side-chains of Lys15,Glu54, and Thr106. The corresponding residues in EcTRPare Leu11, Pro100, Leu101, Leu102, His9, Arg47, andHis98 (see Fig. 1). Whereas the hydrophobicity of the thy-roxine-binding site seems to be maintained, His9 and Arg47lead to a shift in the polarity of the channel. This at least inpart, explains the lack of any thyroxine-binding propertiesin EcTRP (Eneqvist et al., 2003).

3.4. Metal binding to EcTRP

We identiWed 18 metal atoms in the EcTRP electron den-sity map: Wve atoms bound to monomer A and B and fouratoms bound to monomer C and D. These atoms wereconclusively interpreted as zinc ions; they show clear fea-tures in an anomalous diVerence Fourier map, high I/�Ivalues (listed in Table 1), and interact with ligands com-monly known to interact with zinc with tetrahedral geome-tries. Ligand-binding distances also show values close tothose expected for zinc–protein interactions (Table 2) (Har-ding, 2001). However, all zinc-binding sites were not fullyoccupied (Table 1). To further investigate the metal-bind-ing properties of the protein, pure protein samples fromtwo separate puriWcations were analyzed for their contentof metal ions. For one of these, zinc ions were present in themedia during cell growth and protein expression. Onlytrace amounts (<0.01 equivalent) were found in bothprotein samples for all metals assayed: Al, As, Ba, Ca, Cd,Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Pb, S, and Zn. Thusthe protein-bound zinc ions must originate from the 10 mMZnSO4 added to the crystallization medium and necessaryfor crystal growth. This suggests that they might not be anatural ligand for the protein in vivo. Attempts to removebound zinc by back-soaking the crystals in a zinc-free crys-tallization solution rendered the crystals unsuitable fordiVraction studies.


thereby providing additional stability.
Fig. 5. Sulfate binding in the saddle. One sulfate ion binds at each dimer interacting with the side-chain moieties of Trp34 and Arg63 from both monomers,


Intriguingly, however, three of the bound zinc ions arepositioned right at the putative ligand-binding site andinvolve three histidine residues, His9, His96, and His98. Ofthese, His9 and His98 are strictly conserved within the TRPfamily. These two residues bind the central zinc (site I) withoptimal geometry and close to 100% occupancy (Table 1and Fig. 6). At the second zinc-binding site (site II), the zincion interacts with the side chains of residues His96 andHis98. Thus the side chain of His98 can participate in thebinding of two zinc ions. The zinc ion at the second site alsointeracts with the one of the main chain carboxyl oxygensof the conserved residue Ser114 situated at the very C-ter-minal end of the protein. In monomer C, the zinc ion bindsin addition to the carboxyl group of Glu54 from a symme-try-related D monomer. This interaction leads to betterdeWned interaction as suggested by a higher occupancy(Table 1). The zinc ion at site III is only present in mono-mer A and B, where it binds to His96 and to the secondmain chain carboxyl oxygen of Ser114. Overall, the occu-pancies of the zinc ions present at site II and III are low.

The zinc ions at the remaining two sites (sites IV and V)appear to bind less tightly to the protein, which isreXected in the somewhat lower occupancies (Fig. 2). Thezinc ion at site IV binds to the O�1atom of Asp61 and theN�1 atom of His89. In the B- and D-monomers, it alsointeracts with the side chain of Asp31 from symmetry-related D and B monomers. The zinc ion at site Vconnects the side-chains of Glu83 and Glu87. These tworesidues are positioned in the F strands of diVerent mono-mers at the concave side of the saddle, thus metal bindingat this site leads most likely to additional stabilization ofthe dimer.

The C-terminus of EcTRP is shorter than in hTTR. Thelast four residues Tyr111, Arg112, Gly113, and Ser114 forma characteristic Wngerprint for the TRP family. They arepositioned in close vicinity of the putative ligand-bindingsite with its two conserved histidines, His9 and His98. These

Fig. 6. Zinc ion interactions at the putative ligand-binding site of EcTRP in manomalous scatterers in the structure with the Wnal model superimposed. Foucontoured at Wve times the standard deviation, or 5�, for the zinc data (blue) ias red spheres. The interaction involving His9-N�1and His98-N�2 is the majotion geometries. The other two binding partners of the zinc are water moleculthe carboxyl oxygens of the C-terminal residue Ser114. In monomers A and Bforms a hydrogen bond to the carboxyl oxygen of Ser114 rather than bindingdue (¤) also interacts with this zinc ion. It should be noted that the second aTable 1). In monomers A and B, the side chain of His96 is reWned in two orienreader is referred to the web version of this paper.)

residues are likely to play an important role in the functionof EcTRP. At the innermost solvent-excluded parts of theputative ligand-binding cavity, the hydroxyl groups of twoTyr111 residues interact by forming direct hydrogen bonds.These side chains, together with the side chains of twoLeu102 residues, eVectively block the access to the rear partof the channel. In their immediate vicinity, the hydroxylgroups of two Ser109, corresponding to Ser117 in hTTR,form direct hydrogen bonds across the EcTRP monomer-monomer interface. The distance between two Ser109hydroxyl groups across the dimer–dimer interface is 0.8 Åshorter than the corresponding distance across the hTTRinterface (4.5 Å for EcTRP and 5.3 Å for hTTR). As a con-sequence, the channel in EcTRP is not as deep and has abroader entrance than the channel in hTTR. If thyroxine issuperimposed on the EcTRP channel, only the ring struc-ture of its thyrosyl part can be accommodated in the chan-nel, suggesting that putative ligands of EcTRP will mostlikely comprise smaller molecules or molecules restricted toa one-ring structure (Fig. 7). The solvent-excluded area ofthe dimer–dimer interface that creates the channel is2688 Å2 for EcTRP, which is similar to the 2523 Å2 mea-sured for hTTR. However, the electrostatic potential of thechannel contains signiWcantly more positive charges in thecase of EcTRP, which explains its altered ligand-bindingproperties. The main chain of the last three residues Arg112-Ser114 in EcTRP does not fold in a regular �-strand con-formation and the main-chain contacts with �-strand Gpresent in hTTR are missing in EcTRP. Interestingly, theside chain of Arg112 extends in the same direction as themain chain in the hTTR protein. Through a bridging watermolecule, the N� atom of Arg112 forms hydrogen bonds tothe main-chain carbonyl oxygens of Gly113 and His96, andthe main chain amino group of His98. In addition, its N2atom forms hydrogen bonds to the O�2 atom of Glu95 andthe carbonyl oxygen of His96. Therefore, the side chain ofArg112 mimics many of the hydrogen bonds made by the

onomers A, B, C, and D. Experimental electron density maps illustrating therier maps calculated with coeYcients F, �calc ¡90° for the whole molecule

n the P21 structure. Zinc ions are shown as grey spheres and water moleculesr zinc-binding site (site I) as judged from high occupancy and good coordina-es. The zinc ions bound at site II are coordinated by His98, His96, and one of, however, the orientation of His98 is such that the N�1 atom of its side chain to the zinc ion at site II. In the C-monomer a symmetry-related Glu54 resi-nd third zinc-binding sites are not fully occupied in all four monomers (seetations. (For interpretation of the references to color in this Wgure legend, the

Table 2Coordination of Zn2+ at site I in one representative monomer of EcTRP

The values for the bromide soaked structure are shown in brackets. In monomer A the bromide ion replaces water molecule W44.

Distance (Å) Angle (°)

X-Zn-His98 X-Zn-W44(Br1) X-Zn-W45(W45)

His9 N�1 2.1 (2.1) 109.9 (101.6) 119.9 (120.3) 101.13 (105.8)His98 N�2 2.0 (1.9) 107.5 (101.4) 105.05 (113.2)W44 H2O (Br1) 2.2 (2.6) 112.44 (114.0)W45 H2O (W45) 2.3 (2.4)


main chain of �-strand H in hTTR to �-strand G. Fromthis arrangement, we suggest that the function of Tyr111and Arg112 is to provide stability to the EcTRP structure,whereas residues Gly113 and Ser114 could be more impor-tant for the function.

3.5. The zinc-bromide bound form of EcTRP

Mono-valent anions are known inhibitors of manyzinc-enzymes such as carbonic anhydrase and alcoholdehydrogenase. The anions inhibit proper function of theproteins by direct coordination to the active site zinc ion.We have investigated the binding of bromide ions toEcTRP at 2.1 Å resolution. From anomalous diVerenceFourier electron density maps, four bromides (one permonomer) could clearly be identiWed. These were posi-tioned at the primary zinc-binding site, where one of thetwo zinc-bound water molecules was replaced by a bro-mide ion (Fig. 8). Interestingly, the water molecule thatappears to be protected from the bulk solvent by residuesArg47 and Lys49 had been replaced. The anomalous mapof the bromide-bound structure suggests that sulfate ionsbind in the vicinity of the putative ligand-binding site.Their binding is puzzling since they are not present in thezinc-only bound structure even though the crystallizationconditions were identical. In the presence of both zinc andbromide, the side-chain of His98 has shifted its position inall four monomers by about 1 Å. This movement opens upspace in the active site to accommodate the bound sulfate.

No electron density for zinc was observed at site V in thebromide-zinc structure.

In many prokaryotes, the gene for TRP is located withinthe operon encoding proteins that are important for purinedegradation, such as xanthine dehydrogenase, uricase, allan-toinase, and ureidoglycolate hydrolase (Eneqvist et al.,2003). For example, the gene encoding TRP in Bacillus sub-tilis is essential for urate oxidase (uricase) activity (Schultzet al., 2001). Interestingly, two recent studies have indepen-dently shown that TRP facilitates hydrolysis of 5-hydroxy-isourate (HIU), the end product of the uricase reaction (Leeet al., 2005; Ramazzina et al., 2006). From these studies itwas concluded that TRP actually functions as a hydroxyiso-urate hydrolase (HIUH) (Lee et al., 2005; Ramazzina et al.,2006), an enzyme that facilitates the stereo-speciWc conver-sion of HIU to (S)-allantoin (Raychaudhuri and Tipton,2002; Raychaudhuri and Tipton, 2003). HIUH was WrstidentiWed in soybeans where it comprises a protein of 560amino acids homologous to the Family 1 retaining glycosi-dases (Raychaudhuri and Tipton, 2002). Mutational studiesof soybean HIUH and �-glucosidases have shown that, inparticular, two glutamic acid residues situated at the active-site area of the proteins are crucial for activity. The crystalstructure of maize �-glucosidase isozyme 1 (PDB AccessionCode 1ELE, (Czjzek et al., 2001)) shows that these glutamicacid residues are positioned in the vicinity of each other atthe bottom of a smaller ligand-binding cleft.

As pointed out by Lee et al. (Lee et al., 2005) there is nosequence similarity between TRP and soybean HIUH,

Fig. 7. (A) The putative ligand-binding channel of EcTRP and (B) the thyroid hormone-binding channel in hTTR. Thyroxine is shown and superimposedin both structures. The zinc ions at site I are shown as grey spheres and water molecules as red spheres. Due to the close interactions between two Tyr111and two Leu102 residues, the thyroxine molecule cannot be accommodated in the rear part of the EcTRP channel. The hydrophobic channel in EcTRP istherefore shallower and has a broader entrance than the channel in hTTR. The last three residues at the C-terminal end of EcTRP deviate from a regular�-strand structure. For clarity only the C� atom of Ser114 is shown.


suggesting that if the biological role of TRP is to functionas a HIUH, its catalytic mechanism must be diVerent.EcTRP comprises several negatively charged residues, inparticular localized in the saddle area, but these are notconserved within the TRP family (Eneqvist et al., 2003).Neither are there any negatively charged residuespresent in the vicinity of the putative ligand-binding siteof TRP.

It is tempting to speculate that the metal clusters foundin EcTRP are important for function. However, analysisof the metal-content showed that zinc was absent in thepuriWed recombinant protein. Furthermore, zinc was notadded to the buVer used in the HIUH functional study ofB. subtilis TRP (Lee et al., 2005). Further studies areclearly needed to elucidate the proper function of TRP.

Acknowledgments

We thank Therese Eneqvist for helpful discussions andTerese Bergfors for critical reading of the manuscript. Thisstudy was supported by the Swedish Research Science

Council (K2004-03X-13001-06A, K2004-03EF-15196-01A,and K2004-03B-15003-01A), the Gustafsson foundation,the Kempe foundation, and the patients’ associationFAMY/AMYL.

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the transthyretin-related protein: structural investigation of a novel protein family

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