An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma...

doi:10.1006/jmbi.2001.4465 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 271±282

An Unexpected Extended Conformation for the ThirdTPR Motif of the Peroxin PEX5 fromTrypanosoma brucei

Abhinav Kumar1, Claudia Roach1, Irwin S. Hirsh1, Stewart Turley1

SteÂphane deWalque2, Paul A. M. Michels2 and Wim G. J. Hol1*

1Departments of BiologicalStructure and BiochemistryBiomolecular Structure Centerand Howard Hughes MedicalInstitute University ofWashington, SeattleWA 98195, USA2Research Unit for TropicalDiseases (TROP), Christian deDuve Institute of CellularPathology (ICP) and Laboratoryof Biochemistry, UniversiteÂCatholique de Louvain (UCL)Avenue Hippocrate 74B-1200, Brussels, Belgium

E-mail address of the [email protected]

Abbreviations used: TPR, tetratriPTS-1, peroxisomal targeting signal

0022-2836/01/010271±12 $35.00/0

A number of helix-rich protein motifs are involved in a variety of criticalprotein-protein interactions in living cells. One of these is the tetratricopeptide repeat (TPR) motif that is involved, amongst others, in cell cycleregulation, chaperone function and post-translation modi®cations. So far,these helix-rich TPR motifs have always been observed to be a compactunit of two helices interacting with each other in antiparallel fashion.Here, we describe the structure of the ®rst three TPR-motifs of theperoxin PEX5 from Trypanosoma brucei, the causative agent of sleepingsickness. Peroxins are proteins involved in peroxisome, glycosome andglyoxysome biogenesis. PEX5 is the receptor of the proteins targeted tothese organelles by the ``peroxisomal targeting signal-1``, a C-terminal tri-peptide called PTS-1. The ®rst two of the three TPR-motifs of T. bruceiPEX5 appear to adopt the canonical antiparallel helix hairpin structure.In contrast, the third TPR motif of PEX5 has a dramatically different con-formation in our crystals: the two helices that were supposed to form ahairpin are folded into one single 44 AÊ long continuous helix. Such a con-formation has never been observed before for a TPR motif. This raisesinteresting questions including the potential functional importance of a``jack-knife'' conformational change in TPR motifs.

# 2001 Academic Press

Keywords: TPR motif; PEX5; peroxins; trypanosomes
*Corresponding author
Introduction

A number of small compact helix-rich motifshave been discovered in recent years that areinvolved in a wide variety of protein-protein inter-actions. Examples of such helix-rich motifs are thearmadillo repeat consisting of 42 residues foldedinto three helices, the HEAT motif forming twoantiparallel helices out of 37-43 residues, and thetetratrico peptide repeat (TPR) motif comprising 34residues and also forming an antiparallel pair ofhelices (Groves & Barford, 1999). Typically, mul-tiple copies of the same motif occur in tandemrepeats in armadillo and HEAT motif-containingproteins. In general, the sequence conservationamong these motifs is very low, yet, in all struc-tures observed so far, these motifs fold into very

ing author:

co peptide repeat;-1.

similar compact conformations. TPR motifs arecharacterized by a 34-residue degenerate sequence(-W4-L7-G8-Y11-A20-F24-A27-P32-), with signi®cantvariations in the motif-de®ning residues (Sikorskiet al., 1990; Zhang & Grishin, 1999). Here, wereport on a surprisingly new conformation for thestructure of one of the three TPR-motifs from Try-panosoma brucei PEX5, a peroxin involved in glyco-some biogenesis in certain unicellular parasiticorganisms.

Trypanosomatids are protozoa that infect a largenumber of different species, including humans andcattle, causing a wide diversity of diseases speci®-cally in tropical areas. Two subspecies of T. brucei,T. brucei rhodesiense and T. brucei gambiense, areresponsible for the occurrence of the more virulentEast coast and the milder West coast variant ofsleeping sickness, jointly called African trypanoso-miasis (WHO, 1997, 1999). Currently, there is aserious resurgence of sleeping sickness in sub-Saharan Africa while there is a paucity of drugssuitable for treatment of patients in this part of the

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272 Surprising TPR Motif Structure in PEX5

world (Smith et al., 1998; Moore et al., 1999).T. cruzi is related to T. brucei, yet is transmitted bydifferent insect vectors and causes an entirelydifferent disease, called Chagas' disease, whichoccurs in Latin America and is hence also calledAmerican trypanosomiasis. Leishmanias belong tothe same family of protozoa and some 11 speciesof this genus are responsible for a wide spectrumof diseases throughout the tropics, ranging fromself-healing ulcers to horrible facial dis®gurationsand lethal visceral leishmaniasis. There is a despe-rate need for new effective and safe drugs for treat-ment of all of these diseases, particularly in viewof the occurrence of drug resistance (Barrett, 1999).

To this end, we have been studying a uniqueorganelle of these protozoa, called the glycosome.In trypanosomatids, these organelles contain nineenzymes involved in glucose and glycerol metab-olism, in addition to several enzymes responsiblefor b-oxidation, purine metabolism, and other bio-chemical pathways (Opperdoes & Borst, 1977;Opperdoes, 1984; Michels et al., 2000). In spite ofthe unusual feature that the majority of the proteinmass in glycosomes consists of glycolytic enzymes,hence the name of the organelle (Opperdoes &Borst, 1977), the glycosomes from trypanosomatidsshare several key features, and a common origin,with peroxisomes in yeasts and mammals, andwith glyoxysomes in plants (Opperdoes, 1987,1988). This is re¯ected in the similarity in the bio-genesis of these organelles, and speci®cally in theperoxins responsible for importing proteins fromthe cytosol into the organelle matrix of glycosomes,glyoxysomes and peroxisomes (Fung & Clayton,1991; Sommer et al., 1992; Blattner et al., 1995;Flaspohler et al., 1997; de Walque et al., 1999;Jardim et al., 2000; Distel et al., 1996; Brown et al.,2000).

Two glycosomal and peroxisomal import sig-nals have been well characterized (Subramani,1996b). One is PTS-1 (peroxisomal targeting sig-nal-1) that consists of a carboxy-terminal tripep-tide sequence SKL, or variants thereof(Rachubinski & Subramani, 1995; Subramani,1996a). The other is PTS-2, a nonapeptide locatednear, but not at, the N terminus of glycosomaland peroxisomal matrix proteins (Rachubinski &Subramani, 1995; Subramani, 1996a). The PTS-1signal is more common than the PTS-2 signaland is recognized by a receptor protein, PEX5,which is a key protein in mediating import ofPTS-1-containing cargo proteins into the glyco-somes and peroxisomes. PEX5 from severalspecies has been cloned and characterized andappears to be a soluble protein with a molecularmass of approximately 60-70 kDa, that interactsnot only with the cargo proteins, but also witha variety of other peroxins (Elgersma & Tabak,1996; Schliebs et al., 1999). A multiple sequencealignment of PEX5 proteins (Figure 1) reveals acharacteristic C-terminal region comprising sevento eight TPR motifs (Lamb et al., 1995) that areimplicated in binding peroxisomal, glyoxysomal

and glycosomal matrix proteins bearing thePTS-1 signal (Terlecky et al., 1995).

We have determined the crystal structure of the®rst three TPR motifs of T. brucei PEX5 (hereafterreferred to as ``PEX5-T3``) at 1.6 AÊ resolution.Here, we present this structure including a mostunexpected conformation of the third TPR motif.We also discuss the importance of our structure inthe light of the Homo sapiens PEX5 C-terminal TPRdomain structure which was published during thereview process of our paper (Gatto et al., 2000).

Results and Discussion

Overall structure

The selenomethionyl MAD dataset resulted in anexcellent electron density map (Figure 2(a)) thatenabled the construction of essentially a completePEX5-T3 molecule with good geometry and lowR-factors (Table 1). The structure presented hereconsists of 129 residues (14.5 kDa), correspondingto residues 332 to 453 of T. brucei PEX5, with sevenexogenous residues including a His-tag at the Cterminus. The PEX5-T3 molecule has approximatedimensions of 25 AÊ � 30 AÊ � 45 AÊ . Of the 122PEX5-T3 residues, the ®rst two residues at the Nterminus and the last eight residues at the C termi-nus are disordered in the crystal, as are the sevenexogenous C-terminal amino acids. The structureconsists of an 18 residue long N-terminal segmentfollowed by the three TPR motifs that form a right-handed super helix, with the ``A''-helices of theindividual TPR motifs forming the inner concavesurface of the PEX5-T3 super helix. The ®rst twoTPR motifs adopt the canonical TPR fold consistingof a pair of antiparallel a-helices. The third TPR,however, is a single continuous helix, 29 residuesand 44 AÊ in length, which deviates dramaticallyfrom the standard two-helix TPR module. We alsoobserved an Mg2� bound to a key residue of theprotein. Below we look into the three TPR motifs,designated TPR-I, II and III, respectively, after ®rstdescribing the characteristics of the magnesiumbinding site.

Magnesium ion binding

The electron density map of T. brucei PEX5-T3showed the presence of a Mg2� located in a shal-low cavity near the loop connecting helices A2 andB2 of TPR-II and the putative loop within TPR-III(Figure 2(b) and (c)). Consequently, the divalention faces the concave inner groove of the superhelix. The Mg2� is coordinated directly by one ofthe side-chain oxygen atoms of residue E397 andby ®ve water molecules in an octahedral geometry(Figure 2(d)), in a manner strikingly similar to thatobserved in Serratia endonuclease (Miller et al.,1999). Three of these water molecules make hydro-gen bonds to other residues in the same moleculeas well as to two other symmetry-related PEX5-T3molecules. Thus the Mg2� is engaged in promoting

Table 1. Data collection and re®nement statistics

A. Data collection and processingSpace group P65

Unit cell (AÊ ) a�b�75.00, c�56.01Number of molecule/asu 1X-ray source ALSa beam line 5.0.2Anomalous scatterers 4 SeResolution (AÊ ) 20.0-1.6Wavelength (AÊ ) 0.97980 (peak) 0.98000 (infl) 0.95741 (remote)Completenessb (%) 98.1 (99.8) 98.1 (99.7) 97.9 (98.6)I/sI

b 13.8 (3.7) 17.3 (3.6) 17.4 (2.2)Rsym

b (%) 9.1 (48.9) 7.9 (50.2) 8.4 (64.1)

B. Model refinementResolution (AÊ ) 20.0-1.6Rwork/Rfree (%) 19.05/22.26Solvent content in unit cell (%) 67.0Number of protein atoms 871Number of solvent atoms 146Number of Mg2� 1rmsd bond length (AÊ ) 0.02rmsd bond angle (deg.) 1.3Average B-factor of protein atoms (AÊ 2) 20.5 (all) 18.0 (main-chain) 23.0 (side-chain)B-factor of Mg2� (AÊ 2) 17.3Average B-factor of water molecules coordinating

Mg2� (AÊ 2)20.6

Average B-factor of remaining water molecules (AÊ 2) 36.7

a Advanced Light Source synchrotron facility at Berkeley.b Values in parenthesis correspond to the last shell.

Surprising TPR Motif Structure in PEX5 273

crystal contacts, which is well in agreement withthe observation that the presence of Mg2� wascritical in obtaining the crystals. The distances ofthe water molecules and the oxygen atom of E397from the Mg2� range from 1.97-2.14 AÊ , which arewithin the optimal range of magnesium-oxygenligand distances (Poonia & Bajaj, 1979). Of the resi-dues that interact with the Mg2� either directly orthrough water-mediation, residues A394, E397 andD399, are located on the loop between the A and Bhelices of the TPR-II motif. E397, which is the onlydirect protein ligand of the Mg2�, and D399, whichis engaged in a water-mediated interaction withthe magnesium ion, are both either an Asp or aGlu residue in all known PEX5 sequences. Further-more, we note that N429, a residue conserved inall PEX5 sequences, makes H-bonds to D399 in theT. brucei structure, while it is involved in PTS-1 sig-nal peptide binding in the human structure (Gattoet al., 2000).

Intra and inter-TPR motif interactions

The 18 residue long N-terminal segment ofPEX5-T3, comprising a coiled region, a shortb-strand and a four residue 310-helix, wrapsaround the TPR helices on the outside of the superhelix spanning a distance of �32 AÊ . This segmentmakes extensive contacts with the outer face of theTPR super helix (Figure 2(b) and (c)), burying asurface area of �1040 AÊ 2. Most of the interactionsare mediated by hydrogen bonds between proteinatoms directly, although there are a few water-mediated interactions as well. The key residues

linking this segment to the body of the PEX5-T3molecule are N335, Y338, E341, N344, Y346, M347and H349 of the N-terminal segment that interactwith residues N407, E373, E373, L370, E367, K378and E355 of the B helices of the TPR motifs. Ofthese, all residues are highly or absolutely con-served among the known PEX5 sequences exceptresidues N335, E341 and M347. This suggests thatthe conformation of this segment of the PEX5-T3may be conserved in the PEX5 full-length struc-ture. This is substantiated by the comparison of theT. brucei structure with the human structure, whichshows this segment to superimpose within anrmsd value of 0.9 for 16 Ca equivalent atoms.

The helices within as well as between the TPRmotifs interact mainly through hydrophobic resi-dues. The helices A and B bury a surface area of�374 AÊ 2 and �486 AÊ 2 within TPR-I and TPR-II,whereas helices B and A of adjacent TPR motifsbury a surface area of �506 AÊ 2 and �464 AÊ 2 forthe TPR-I:TPR-II and TPR-II:TPR-III interfaces,respectively. Within TPR-I, the A and B helicespack against each other through highly conservedresidues including G356, L360, A363, L365, E367,A371, and F372 (Figure 1). Of these, residues G356and L365 are completely conserved amongst allPEX5s. The packing interactions within TPR-II aregenerally very similar as in TPR-I with hydro-phobic and highly conserved residues mediatingthe inter-helical interactions. Among these, resi-dues W386, G390 and N396 are invariant, andA394, E397, A402, H408, and L412 highly con-served among the PEX5 sequences. Inter-TPR pack-ing involving the helices B and A of adjacent TPR

Figure 1. A multiple sequence alignment of PEX5 from various species. Five out of over a dozen sequences, corre-sponding to T. brucei (labeled TB, TrEMBL accession number Q9U7C3), H. sapiens (labeled HS, SWISS-PROT accessionnumber P50542), Mus musculus (labeled MM, SWISS-PROT accession number O09012), Saccharomyces cerevisiae(labeled SC, SWISS-PROT accession number P35056), and Citrullus lanatus (labeled CL,TrEMBL accession numberO81280) PEX5, have been included in the Figure in order to limit its size while including representatives from widelydiverse species. The alignment was performed with CLUSTALW (Thompson et al., 1994) and formatted by ALSCRIPT(Barton, 1993). The sequence numbering refers to the T. brucei sequence. Completely conserved amino acids aredepicted in red, substitution with similar residues in blue, non-conserved residues in black, while gaps are shown asdots. The secondary structure elements in PEX5-T3 are indicated above the sequence. The overall sequence identity ofT. brucei PEX5 with PEX5s from different species ranges from 22-27 %. The C-terminal region containing the TPRmotifs exhibits 31-40 % identity (de Walque et al., 1999).


motifs is again mediated essentially by hydro-phobic residues. Six out of a total of 18 inter-TPRcontact residues are absolutely conserved in allsequences of PEX5. It is likely that the arrangementof the ®rst ®ve helices of T. brucei PEX5-T3 is con-served in the PEX5 family in view of the highdegree of conservation of the interacting residues.However, the third TPR motif of T. brucei PEX5displays a conformation never seen before for TPRmotifs.

The third TPR motif

In the present crystal structure the TPR-III motifforms a single extended helix, which is entirelydifferent from the canonical TPR-fold. This resultsin the Ca atom of the last observed residue L445

being about 41 AÊ away from the expected positionhad TPR-III adopted the canonical fold. This strik-ing difference is accompanied by a surprising con-formation for the tripeptide E430-H431-N432 thatwas expected to form a turn between residues 416-429 (referred to as the ``A3`` helix in this paper)and residues 433-445 (the ``B3`` helix). However,instead of forming a turn, residues 430-432 areseen to adopt a perfectly helical conformation,thereby bridging A3 and B3 helices into a continu-ous helical structure for this TPR-motif.

This is the ®rst time that such an extended con-formation for a TPR-motif has been observed andraises at least two sets of intriguing questions. Oneset is related to fundamental issues of protein fold-ing: can TPR motifs adopt such an extended all-helical conformation in general? Or is the third

Figure 2. Electron density map, the overall structure, and the Mg2� binding site of T. brucei PEX5-T3. (a) Stereo viewof a typical region in the 2Fo ÿ Fc map contoured at 1s above the mean with the re®ned model of the protein superim-posed in the map. (b) The overall structure of PEX5-T3. The N-terminal coil region and all the loop regions in the struc-ture are shown in gold, while the three TPR motifs are depicted in purple, green and fuschia, respectively. The A and Bhelices in each TPR as well as the Mg ion contacting residue E397 have been labeled. The third TPR motif does not con-form to the standard two-helix TPR motif, but exists as a continuous long helix. The putative loop region connecting thetwo helices in TPR-III is depicted in gold. (c) A 90 � rotated view of the structure shown in (b). The B3 helix of TPR-IIImotif from a symmetry-related molecule is added to show its position with respect to helices A1, A2 and A3. (d) A stereoview of the Mg2� binding site with the Mg2� (in green) coordinated by ®ve water molecules (numbered 1-5 and in red)and the side-chain OE atom of residue E397. Residues making hydrogen bonds (depicted by dotted lines) in the networkof interactions around the Mg2� are also included. The helices in green, fuschia and purple belong to three different mol-ecules related by crystallographic symmetry. Figure (a) was made by BobScript (Esnouf, 1999), while (b)-(d), Figures 3and 4 were made by MolScript (Kraulis, 1991). All of these Figures were rendered by Raster3D (Merritt & Bacon, 1997).


Figure 3 (legend opposite)


TPR motif of T. brucei PEX5 a special case? Is theconformation observed in the PEX5-3T crystalsinduced by crystal contacts, and hence rare in sol-ution, or has our crystal captured a fold that doesoccur to a signi®cant extent in solution as well?

A second group of questions is more closelyrelated to the function of TPR motifs, and of PEX5in particular. Is the extended conformation of TPR-III conserved in full-length PEX5? Will thisextended conformation be maintained in full-length PEX5 while this peroxin forms complexeswith other proteins? Does PEX5 TPR-III undergo a``jack-knife'' motion during a membrane transloca-tion step? A broader question is: would certainTPR motifs in other proteins utilize this dramaticopen-and-close conformational motion while thoseproteins perform their functions?

Obviously answering such broad biologicalquestions is outside the realm of this report that

focuses on the crystal structure of T. brucei PEX5-T3, but an analysis of our structure of TPR-III canset the stage for further investigations. The struc-ture of TPR-III is not only remarkable in that itshows a fully extended conformation, but also inthat the two hydrophobic surfaces from the ``A3``and the ``B3`` helices do not engage in interactionswith other helices that mimic the canonical A3-B3interactions. Instead, the putative A3 and B3helices engage in a complex set of interactions withhelices from neighboring molecules. These inter-actions involve helices arranged in a perpendicularorientation (Figure 3(a)) that is completely differentfrom the antiparallel A-B helix interaction patternwithin a canonical TPR motif. Speci®cally, the B3helix makes over 20 contacts with helices A1, A2,and A3 of a symmetry-related molecule such thatthe axis of the B3 helix runs perpendicular to thoseof the three interacting A helices (Figure 3(a) and

Figure 3. Conformation and interactions of the T. brucei TPR-III motif. (a) The extended conformation of TPR-IIIhelix (in fuschia) with the loop connecting its two helices, A3 and B3, in gold. Three A helices of a neighboring mol-ecule interacting with B3 are depicted in green. Crystal contacts with A3 involve a loop in the N-terminal region (notshown in the Figure) and the TPR-III helix (shown in red) from two different molecules. The contact residues of A3and B3 that would interact with each other in a canonical TPR fold are colored blue for A3 and red for B3. (b) Left-hand panel: Helix B3 depicted in light fuschia with its contact residues in red cpk and the interacting residues of thesymmetry related molecules in the crystal in green. Right-hand panel: Helix B3N derived from the modeled canonicalTPR-III motif (on the basis of the human PEX5 TPR domain structure reported by Gatto (2000) depicted in lightfuschia with its contact residues in red cpk and the interacting residues of helix A3 in blue. (c) Left-hand panel: HelixA3 depicted in dark fuschia with its contact residues in blue cpk and the interacting residues of the symmetry relatedmolecules in the crystal in red. Right-hand panel: Helix A3 depicted in dark fuschia with its contact residues in bluecpk and the interacting residues of helix B3N from the modeled canonical TPR-III motif in red.



(b)). Further interactions with B3 are mediated by aloop near the beginning of the TPR-I motif ofanother symmetry-related molecule. The A3 helixcontacts two segments of a third neighboringmolecule, including the turn between helices A2and B2 of TPR-II and two residues of helix A3. Thecrystal contacts of A3 and B3 bury a total surfacearea of �914 AÊ 2.

An analysis of the structures of 14 canonical TPRmotifs (Das et al., 1998; Gatto et al., 2000; Scheu¯eret al., 2000; TPR-I and TPR-II described here)shows that, on average, the buried surface in aTPR motif due to contacts between the A and Bhelices is 426 AÊ 2. This is signi®cantly less than the�914 AÊ 2 buried by the three symmetry-relatedmolecules interacting with the extended A3-B3helix. If we make the assumption that buried sur-face areas are proportional to the free energies ofinteraction, then the crystal contacts are more thantwice as favorable as the contacts within a typicalTPR fold. Interestingly, the intra-TPR contact sur-face of the B3 helix (red residues in Figure 3(b)) ishardly involved in any crystal contacts in theT. brucei TPR-III motif and appears to be solventexposed while other residues of the B3 helix aremaking contacts with A3 helices from neighboringmolecules. The A3 intra-TPR contact surface (bluein Figure 3(c)) is engaged in a limited number ofcrystal contacts. Evidently, the A3 and B3 heliceshave multiple ways of forming intra or inter-mol-ecular interactions with other proteins.

Comparison with TPR motifs fromother proteins

A comparison of T. brucei PEX5-T3 with otherproteins using the Dali server (Holm & Sander,1993) clearly highlights the similarities and differ-ences among TPR-containing proteins (Figure 4).The Dali search returned a list of several proteinscontaining TPR motifs, with the Hop-TPR1 domain(Scheu¯er et al., 2000), which contains three TPRmotifs, being most similar to the ®rst ®ve helices ofT. brucei PEX5-T3 with a Z-score of 11.4 and anrmsd of 2.3 AÊ between 85 equivalent Ca atoms.The next two most similar proteins found were theTPR domain of serine-threonine protein phospha-tase 5 (Das et al., 1998) and the TPR2A domain ofHop. Overall, ®ve helices from these domains arevery similar to T. brucei PEX5-T3, but differ entirelyin the folding of the third TPR motif since TPR-IIIis unique in showing a single continuous helix inour T. brucei PEX5-T3 structure.

Comparison with human PEX5

After the reviews of the current manuscript werereceived, the study by Gatto et al. (2000) appearedshowing that TPR-III of human PEX5 can adoptthe canonical compact 3TPR fold. A comparison ofT. brucei PEX5-T3 with the ®rst three TPR motifs ofhuman PEX5 reveals that the ®rst ®ve helicessuperimpose within 0.64 AÊ for 78 Ca atoms, with

the B3 helix, of course, completely deviating. Giventhe 46 % sequence identity of the TPR-III motifs inhuman and T. brucei PEX5, and the likelihood thatthe functioning of PEX5 in peroxisomal andglycosomal biogenesis is very similar, TPR-III ofT. brucei can probably also adopt the canonicalcompact TPR conformation. Whether or not full-length PEX5 s can adopt the ``open'' conformationobserved in T. brucei TPR-III, and what the role ofsuch a conformational change would be in peroxi-some, glyoxysome and glycosome biogenesisremains to be established. The TPR ®ngerprintsequences of the T. brucei and H. sapiens TPR-IIImotifs are, respectively: -H4-L7-A8-H11-A20-L24-W27-P32- and -L4-L7-A8-F11-A20-L24-W27-P32. Both ®nger-prints differ somewhat from the canonical ®nger-print derived by Zhang & Grishin (1999), as givenin the Introduction, but not more so than severalother TPR sequences which have been observed toadopt the canonical TPR structure (Sikorski et al.,1990; Zhang & Grishin, 1999). The major differencein residue size between PEX5 TPR-III and otherTPR folds is observed at position 27 of the ®nger-print where the canonical Ala residue is replacedby a Trp residue which is completely conserved inall known PEX5 TPR-III sequences (Figure 1). ThisTrp side-chain appears to be accommodated by thecanonical hairpin conformation of H. sapiens TPR-III without problems, since this TPR-III superim-poses within 0.5 to 0.7 AÊ rmsd for 28 Ca positionsonto TPR-I and TPR-II in H. sapiens PEX5. How-ever, this similarity in conformation of the compactform of TPR-III to other TPR-hairpins does not pro-vide any information on the energy barriers whichTPR-III has to overcome to adopt a fully extendedconformation as seen in the T. brucei PEX5 TPR-IIIstructure.

Clearly, further studies are needed to establishwhich predominant conformation is adopted byTPR-III in the T. brucei PEX5-T3 protein in solution,and which in the full-length PEX5 protein duringglycosome biogenesis. The latter question is mostcomplex to answer fully, since PEX5 engages ininteractions with numerous other proteins includ-ing not only the PTS-1 containing cargo proteins,but also other peroxins, such as PEX13, PEX14 andPEX17 (Huhse et al., 1998; Schliebs et al., 1999;Urquhart et al., 2000). Moreover, PEX5 may interactwith the peroxisomal and glycosomal membranebilayer, in particular if the proposed cytosol-per-oxisome recycling mode of action of PEX5 is oper-ative in T. brucei (Terlecky & Fransen, 2000). Inaddition, Gatto et al. (2000) note that it has notbeen possible to obtain crystals of the H. sapiensTPR domain of PEX5 in the absence of a boundpeptide, which might indicate signi®cant ¯exibilityin this domain. This ¯exibility might re¯ect a capa-bility of TPR-III to adopt a variety of confor-mations, of which we now may have seen twoextremes: the compact and the fully extendedforms. A jack-knife motion of TPR-III could be cru-cial for PEX5 in interacting with other peroxins.Structures of apo-PEX5 and of binary and ternary

Figure 4. A structural superposition of T. brucei PEX5-T3 with other TPR-containing proteins. The views in theright panel are rotated �90 � with respect to those in the left-hand panel. The T. brucei PEX5-T3 molecule in each rowis rendered in fuschia while the structurally similar proteins (PP5 in top row (A,B), Hop-TPR1 in middle (C,D), andHop-TPR2A in bottom row (E,F)) are drawn in green. The Mg2� in PEX5-T3 is shown in red in each panel and thepeptides complexed with the Hop structures are shown in cyan.


complexes of PEX5 with other peroxins will be ofgreat interest in answering this question. Moregenerally, our studies imply that certainTPR-motifs in other proteins may also be able to

jack-knife, with possibly important functionalimplications.

We do, ®nally, like to point out that the positionof the Mg2� observed in T. brucei PEX5-T3 falls


essentially on top of Cd of R520 in the humanstructure. Clearly Mg2� binding at this position inhuman PEX5 would preclude R520 to assume theconformation observed in the human PEX5-penta-peptide complex. Interestingly, as mentioned ear-lier, the residue which is a direct ligand of the ion,E397 in T. brucei PEX5-T3 (Figure 2(d)), is con-served as either a Glu or Asp residue in all knownPEX5 sequences. Whether or not Mg2� plays anyrole in the functioning of PEX5, e.g. by promotingthe release of cargo proteins into peroxisomes,glyoxysomes and/or glycosomes, needs to befurther investigated.

Materials and Methods

The DNA segment corresponding to the ®rst threeTPR motifs of T. brucei PEX5-T3 was subcloned by PCRusing the full-length gene of PEX5 as template and twoprimers: (1) the forward primer 50-GCAGGAGCATATG-CAGAACAACAC-30, and (2) the reverse primer50-GGTTAACTGACTCGAGTTGTTCATAC-30. The PCRproduct was integrated into the pET26b vector (Nova-gen) at the NdeI and XhoI cloning sites. The resultantplasmid was transformed into Escherichia coli BL21 (DE3)cells. The PEX5-T3 protein was expressed by inductionwith IPTG (1 mM) at 18 �C for eight hours and puri®edby Ni-NTA af®nity chromatography column (Qiagen)utilizing the (His)6 tag fused at the C terminus of theprotein. The puri®ed protein was dialyzed against aphosphate buffer (20 mM, pH 7.0) containing 100 mMNaCl and concentrated to about 20 mg/ml. Forexpression of Se-Met protein, the cells were grown andexpressed in M9 minimal media supplemented withamino acids (100 mg each of Lys, Thr, and Phe, and50 mg each of Val, Leu, Ile, and Se-Met in 1 liter culture)and glucose (0.4 % w/v) as described by Van Duyne et al.(1993).

The crystals of PEX5-T3 were grown by the sittingdrop vapor diffusion method. Equal volumes of the pro-tein and reservoir solution (2 ml of each) were mixed andallowed to equilibrate against the reservoir solution con-taining 100 mM imidazole (pH 6.5), 50 mM MgCl2,5 mM IPTG and 5 % (v/v) DMSO at 20 �C. Small rod-like hexagonal crystals appeared within a day of settingup the crystal tray which grew to a size of 10 mm �10 mm � 50 mm within four to ®ve days. A crystal wastransferred from the original sitting drop to a cryoprotec-tant solution containing 22 % glycerol in the reservoirsolution, allowed to soak for �30 seconds and then¯ash-frozen in liquid nitrogen before being exposed tothe X-ray beam in a cold nitrogen stream. A MAD dataset was collected at the ALS synchrotron (beam line5.0.2) from a crystal grown with the Se-Met protein. Thedata were collected at three wavelengths (correspondingto the peak, in¯ection, and a remote point on the SeEXAFS spectrum) with a CCD detector. The data wereprocessed and scaled with the HKL software package(Otwinowski & Minor, 1997).

The four Se atom positions were determined, re®nedand initial phases were calculated by the programSOLVE (Terwilliger & Berendzen, 1999) in two enantio-meric space groups P61 and P65. The Se-atom phasedmaps immediately established the space group to be P65

due to the recognizable features in the electron densitymap in this latter space group. The initial phases werere®ned by wARP (Perrakis et al., 1999) to produce a com-

pletely interpretable map with about 95 % of the modelof the protein built into the map. The model was checkedand completed manually using the program O (Joneset al., 1991). The model was re®ned with REFMAC(Murshudov et al., 1999) of the CCP4 software package(Bailey, 1994) to a ®nal R-factor of 19.0 % and an Rfree of22.3 %. The re®ned model contains 112 protein residues(the ®rst two N-terminal and the last 15 C-terminal resi-dues being disordered), 146 water molecules and onemagnesium ion. The geometric quality of the model waschecked by the program PROCHECK (Laskowski et al.,1993) which showed all residues to lie within allowed orgenerously allowed regions of the Ramachandran plot.The data collection and re®nement statistics are providedin Table 1.

Protein Data Bank accession code

The coordinates have been deposited in the RCSB,with accession code 1HXI.

Acknowledgments

We thank Drs Gerry McDermott at ALS synchrotronfor help with the X-ray data collection, Stephen Sureshfor many helpful discussions during the structure deter-mination and re®nement of the model, Francis Athap-pilly and Ethan Merritt for maintaining our computingenvironment, Brad Ornstein for assistance with proteinpuri®cation and Christophe Verlinde for stimulating dis-cussions. W.H. acknowledges a major equipment grantto the Biomolecular Structure Center by the MurdockCharitable Trust.

References

Bailey, S. (1994). The CCP4 suite-programs for proteincrystallography. Acta Crystallog. sect. D, 50, 760-763.

Barrett, M. P. (1999). The fall and rise of sleeping sick-ness. Lancet, 353, 1113-1114.

Barton, G. J. (1993). ALSCRIPT: a tool to format multiplesequence alignments. Protein Eng. 6, 37-40.

Blattner, J., DoÈrsam, H. & Clayton, C. E. (1995). Functionof N-terminal import signals in trypanosome micro-bodies. FEBS Letters, 360, 310-314.

Brown, T. W., Titorenko, V. I. & Rachubinski, R. A.(2000). Mutants of Yarrowia lipolytica PEX23 geneencoding an integral peroxisomal membrane per-oxin mislocalize matrix proteins and accumulatevesicles containing peroxisomal matrix and mem-brane proteins. Mol. Biol. Cell, 11, 141-152.

Das, A. K., Cohen, P. W. & Barford, D. (1998). Thestructure of the tetratricopeptide repeats of proteinphosphatase 5: implications for TPR-mediatedprotein-protein interactions. EMBO J. 17, 1192-1199.

de Walque, S., Kiel, J. A., Veenhuis, M., Opperdoes, F. R.& Michels, P. A. (1999). Cloning and analysis of thePTS-1 receptor in Trypanosoma brucei. Mol. Biochem.Parasitol. 104, 106-119.

Distel, B., Erdmann, R., Gould, S. J., Blobel, G., Crane,D. I., Cregg, J. M., Dodt, G., Fujiki, Y., Goodman,J. M., Just, W. W., Kiel, J. A., Kunau, W. H.,Lazarow, P. B., Mannaerts, G. P. & Moser, H. W.,et al. (1996). A uni®ed nomenclature for peroxisomebiogenesis factors. J. Cell. Biol. 135, 1-3.


Elgersma, Y. & Tabak, H. F. (1996). Proteins involved inperoxisome biogenesis and functioning. Biochim.Biophys. Acta, 1286, 269-283.

Esnouf, R. M. (1999). Further additions to MolScriptversion 1.4, including reading and contouring ofelectron-density maps. Acta Crystallog. sect. D, 55,938-940.

Flaspohler, J. A., Rickoll, W. L., Beverley, S. M. &Parsons, M. (1997). Functional identi®cation of aLeishmania gene related to the peroxin 2 genereveals common ancestry of glycosomes and peroxi-somes. Mol. Cell. Biol. 17, 1093-1101.

Fung, K. & Clayton, C. (1991). Recognition of a peroxi-somal tripeptide entry signal by the glycosomes ofTrypanosoma brucei. Mol. Biochem. Parasitol. 45, 261-264.

Gatto, G. J. J., Geisbrecht, B. V., Gould, S. J. & Berg, J. M.(2000). Peroxisomal targeting signal-1 recognitionby the TPR domains of human PEX5. Nature Struct.Biol. 7, 1091-1095.

Groves, M. & Barford, D. (1999). Topological character-istics of helical repeat proteins. Curr. Opin. Struct.Biol. 9, 383-389.

Holm, L. & Sander, C. (1993). Protein structure compari-son by alignment of distance matrices. J. Mol. Biol.233, 123-138.

Huhse, B., Rehling, P., Albertini, M., Blank, L., Meller,K. & Kunau, W. H. (1998). Pex17p of Saccharomycescerevisiae is a novel peroxin and component of theperoxisomal protein translocation machinery. J. CellBiol. 140, 49-60.

Jardim, A., Liu, W., Zheleznova, E. & Ullman, B. (2000).Peroxisomal targeting signal-1 receptor proteinPEX5 from Leishmania donovani. J. Biol. Chem. 275,13637-13644.

Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M.(1991). Improved methods for building proteinmodels in electron density maps and the location oferrors in these models. Acta Crystallog. sect. A, 47,110-119.

Kraulis, P. (1991). MOLSCRIPT: a program to produceboth detailed and schematic plots of protein struc-tures. J. Appl. Crystallog. 24, 946-950.

Lamb, J. R., Tugendreich, S. & Hieter, P. (1995). Tetra-trico peptide repeat interactions: to TPR or not toTPR? Trends Biochem. Sci. 20, 257-259.

Laskowski, R. A., MacArthur, M. W., Moss, D. S. &Thornton, J. M. (1993). PROCHECK: a program tocheck the stereochemical quality of protein struc-tures. J. Appl. Crystallog. 26, 283-291.

Merritt, E. A. & Bacon, D. J. (1997). Raster3D: photo-realistic molecular graphics. Methods Enzymol. 277,505-524.

Michels, P. A., Hannaert, V. & Bringaud, F. (2000).Metabolic aspects of glycosomes in trypanosomati-dae - new data and views. Parasitol. Today, 16, 482-489.

Miller, M. D., Cai, J. & Krause, K. L. (1999). The activesite of Serratia endonuclease contains a conserved.J. Mol. Biol. 288, 975-987.

Moore, A., Richer, M., Enrile, M., Losio, E., Roberts, J. &Levy, D. (1999). Resurgence of sleeping sickness inTambura County, Sudan. Am. J. Trop. Med. Hyg. 61,315-318.

Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson,K. S. & Dodson, E. J. (1999). Ef®cient anisotropicre®nement of macromolecular structures using FFT.Acta Crystallog. sect. D, 55, 247-255.

Opperdoes, F. R. (1984). Localization of the initial stepsin alkoxyphospholipid biosynthesis in glycosomes(microbodies) of Trypanosoma brucei. FEBS Letters,169, 35-39.

Opperdoes, F. R. (1987). Compartmentation of carbo-hydrate metabolism in trypanosomes. Annu. Rev.Microbiol. 41, 127-151.

Opperdoes, F. R. (1988). Glycosomes may provide cluesto the import of peroxisomal proteins. TrendsBiochem. Sci. 13, 255-260.

Opperdoes, F. R. & Borst, P. (1977). Localization of nineglycolytic enzymes in a microbody-like organelle inTrypanosoma brucei: the glycosome. FEBS Letters, 80,360-364.

Otwinowski, Z. & Minor, W. (1997). Processing X-raydiffraction data collected in oscillation mode.Methods Enzymol. 276, 307-326.

Perrakis, A., Morris, R. & Lamzin, V. S. (1999).Automated protein model building combined withiterative structure re®nement. Nature Struct. Biol. 6,458-463.

Poonia, N. S. & Bajaj, A. (1979). Coordination chemistryof alkali and alkaline earth cations. Chem. Rev. 79,389-445.

Rachubinski, R. A. & Subramani, S. (1995). How pro-teins penetrate peroxisomes. Cell, 83, 525-528.

Scheu¯er, C., Brinker, A., Bourenkov, G., Pegoraro, S.,Moroder, L., Bartunik, H., Hartl, F. U. & Moare®, I.(2000). Structure of TPR domain-peptide complexes:critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell, 101, 199-210.

Schliebs, W., Saidowsky, J., Agianian, B., Dodt, G.,Herberg, F. W. & Kunau, W. H. (1999). Recombi-nant human peroxisomal targeting signal receptorPEX5. Structural basis for interaction of PEX5 withPEX14. J. Biol. Chem. 274, 5666-5673.

Sikorski, R. S., Boguski, M. S., Goebl, M. & Hieter, P.(1990). A repeating amino acid motif in CDC23de®nes a family of proteins and a new relationshipamonb genes required for mitosis and RNA syn-thesis. Cell, 60, 307-317.

Smith, D. H., Pepin, J. & Stich, A. H. (1998). HumanAfrican trypanosomiasis: an emerging public healthcrisis. Br. Med. Bull. 54, 341-355.

Sommer, J. M., Cheng, Q. L., Keller, G. A. & Wang, C. C.(1992). In vivo import of ®re¯y luciferase into theglycosomes of Trypanosoma brucei and mutationalanalysis of the C-terminal targeting signal. Mol.Biol. Cell, 3, 749-759.

Subramani, S. (1996a). Convergence of model systemsfor peroxisome biogenesis. Curr. Opin. Cell Biol. 8,513-518.

Subramani, S. (1996b). Protein translocation into peroxi-somes. J. Biol. Chem. 271, 32483-32486.

Terlecky, S. R., Nuttley, W. M., McCollum, D., Sock, E.& Subramani, S. (1995). The Pichia pastoris peroxi-somal protein PAS8p is the receptor for theC-terminal tripeptide peroxisomal targeting signal.EMBO J. 14, 3627-3634.

Terwilliger, T. C. & Berendzen, J. (1999). AutomatedMAD and MIR structure solution. Acta Crystallog.sect. D, 55, 849-861.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).CLUSTAL W: improving the sensitivity of progress-ive multiple sequence alignment through sequenceweighting, position-speci®c gap penalties andweight matrix choice. Nucl. Acids Res. 22, 4673-4680.

Urquhart, A. J., Kennedy, D., Gould, S. J. & Crane, D. I.(2000). Interaction of Pex5p, the type 1 peroxisome


targeting signal receptor, with the peroxisomalmembrane proteins Pex14p and Pex13p. J. Biol.Chem. 275, 4127-4136.

Van Duyne, G. D., Standaert, R. F., Karplus, P. A.,Schreiber, S. L. & Clardy, J. (1993). Atomic struc-tures of the human immunophilin FKBP-12 com-plexes with FK506 and rapamycin. J. Mol. Biol. 229,105-124.

WHO (1997). Tropical Disease Research: Progress 1995-1996, Thirteenth Programme Report, World HealthOrganization, Geneva.

WHO (1999). Tropical Disease Research: Progress 1997-1998, Fourteenth Programme Report, World HealthOrganization, Geneva.

Zhang, H. & Grishin, N. V. (1999). The a-subunit ofprotein prenyltransferases is a member of the tetra-tricopeptide repeat family. Protein Sci. 8, 1658-1667.

Edited by D. Rees

(Received 16 October 2000; received in revised form 12 January 2001; accepted 12 January 2001)

An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma...

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Transcript of An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma...