The structural and functional diversity of Hsp70 proteins ... · Since chaperones constitute one of the major protein families in the parasitophorous vacuole in an infected erythrocyte

10.1110/ps.072918107Access the most recent version at doi: 2007 16: 1803-1818 Protein Sci.

Addmore Shonhai, Aileen Boshoff and Gregory L. Blatch

Plasmodium falciparumThe structural and functional diversity of Hsp70 proteins from

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REVIEW

The structural and functional diversity of Hsp70 proteinsfrom Plasmodium falciparum

ADDMORE SHONHAI, AILEEN BOSHOFF, AND GREGORY L. BLATCHDepartment of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa

Abstract

It is becoming increasingly apparent that heat shock proteins play an important role in the survival ofPlasmodium falciparum against temperature changes associated with its passage from the cold-bloodedmosquito vector to the warm-blooded human host. Interest in understanding the possible role ofP. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes.Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) andits endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from thestructural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There isincreasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle ofP. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40partners are implicated in the intracellular and extracellular trafficking of proteins. This reviewsummarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, takingadvantage of inferences gleaned from their structures and information on their cellular localization. Thepossible associations between P. falciparum Hsp70s with their cochaperone partners as well as otherchaperones and proteins are discussed.

Keywords: PfHsp70; heat shock protein; Hsp40; molecular chaperone; malaria

Heat shock proteins are highly conserved, ubiquitousproteins that occur in most life forms, whose main roleis to act as molecular chaperones. As molecular chaper-ones, heat shock proteins bind to nonnative proteins,facilitating their refolding to the native state (Ellis 1987).Heat shock protein 70 (Hsp70) forms one of the majorheat shock protein families. Generally Hsp70 proteinsare induced in response to stress, although some Hsp70species are constitutively expressed in cells. Hsp70 bindsto peptide substrate, allowing it to refold, followed byrelease of the substrate in ATP-expending cycles (Szaboet al. 1994). In the ADP-bound state, Hsp70 has high

affinity for the peptide substrate, while its affinity for thesubstrate is reduced in the ATP-bound state (Suh et al.1999). ATP binding induces a conformational change thattranscends to the peptide-binding domain from theATPase domain, resulting in the protein attaining a lowsubstrate affinity status, leading to release of substrate(Liberek et al. 1991). Therefore, nucleotide exchange isessential for the Hsp70 functional cycle to proceed. Tothis end, Bcl-2-associated athanogene (Bag-1) (Hohfeldand Jentsch 1997) and Hsp70-binding protein (HspBP1)(Kabani et al. 2002) have been identified as the nucleo-tide exchange factors (NEFs) for mammalian Hsp70s,while GrpE facilitates nucleotide exchange by DnaK, thebacterial Hsp70 homolog (Harrison et al. 1997). Besidestheir role in refolding nascent proteins, Hsp70s partic-ipate in several other processes in the cell such as theassembly or disassembly of multiprotein complexes(Song et al. 2005), protein translocation (Gambill et al.

Reprint request to: Gregory L. Blatch, Department of Biochemistry,Microbiology and Biotechnology, Rhodes University, Grahamstown6140, South Africa; e-mail: [email protected]; fax: 27-46-622-3984.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072918107.

Protein Science (2007), 16:1803–1818. Published by Cold Spring Harbor Laboratory Press. Copyright � 2007 The Protein Society 1803



1993), protein degradation (Bercovich et al. 1997), signaltransduction (Asea et al. 2002), and prion replication(Song et al. 2005). Hsp70 proteins have a molecular massof ;70 kDa and consist of two distinct domains; the 45-kDa N-terminal domain that binds ATP, and the 25-kDapeptide-binding domain (Flaherty et al. 1990; Wang et al.1993). Hsp70 proteins are localized in the Escherichiacoli cytosol and all compartments of eukaryotic cells suchas chloroplast, endoplasmic reticulum lumen, mitochon-drial matrix, and the cytosol (Yalovsky et al. 1992;Johnson and Craig 1997).

Approximately 3 billion people live in malaria endemicareas, and at least 1 million of these die from the disease(World Health Organization 2005). The following fourspecies of Plasmodium cause malaria: Plasmodium falcipa-rum, Plasmodium vivax, Plasmodium malaria, and Plasmo-dium ovale. Of these, it is P. falciparum that accounts for thehighest mortality rate (Thiel 2005). Therefore, it is falcipa-rum malaria that has received a lot of research attention. Theproduction and localization of inducible Hsp70 proteins inP. falciparum have been well documented (Kumar et al.1991; Joshi et al. 1992; Biswas and Sharma 1994). Theoverproduction of certain isoforms of Hsp70 proteins hasbeen complemented by confirmation of the production ofantibodies against these proteins in human subjects living inmalaria endemic areas (Kumar et al. 1990; Behr et al. 1992).

There is growing evidence that heat shock proteinsfrom P. falciparum could play a cytoprotective role in thelife cycle of the parasite. The importance of chaperones atthe host–parasite interface as a survival strategy has beenreviewed (Feder and Hofmann 1999). Because the lifecycle of P. falciparum transcends across two habitats (thecold-blooded mosquito vector and the warm-bloodedhuman host), it is thought that the production of chaper-ones by the parasite in response to stress is a survivalstrategy against temperature and physiological changesexperienced by the parasite (Sharma 1992). During thedevelopment of febrile malaria, body temperature rises to41°C because of the release of pro-inflammatory cytokinetumor necrosis factor (Karnumaweera et al. 1992), andthe development of this fever is known to promote thepathogenesis of malaria by enhancing the ability of theparasite-infected erythrocytes to adhere to blood vessels(Udomsangpetch et al. 2002). Another study establishedthat P. falciparum parasites that were initially exposed toheat shock were not only able to mount better heat resis-tance to subsequent heat treatments but also showed bettersurvival resilience and improved infectivity (Pavithra et al.2004). It has also been proposed that febrile episodesthat mark the development of malaria also promote theintra-erythrocytic development of the parasite (Pavithraet al. 2004). This phenomenon has been observed in otherprotozoan species (Soete et al. 1994; Wiesgigl and Clos2001).

Pathogenic organisms produce heat shock proteinsduring the invasion of host cells (Maresca and Kobayashi1994; Zugel and Kaufmann 1999), and as a result, heatshock proteins have become targets of vaccine research(Newport 1991). Because of their ubiquity and conserva-tion, it has been proposed that heat shock proteins areat the interface between infection and autoimmunitythrough recognition of conserved epitopes or throughcross-reactivity (Zugel and Kaufmann 1999). In addition,there is evidence that links heat shock proteins with drugresistance, and both heat shock protein 90 (Hsp90) andits functional partner calcineurin have been implicated(Sanglard et al. 2003; Cowen and Lindquist 2005).

Since chaperones constitute one of the major proteinfamilies in the parasitophorous vacuole in an infectederythrocyte (Fig. 1; Nyalwidhe and Lingelbach 2006),they could play an important role in the traffickingof proteins of parasitic origin into the erythrocyte.

Figure 1. Cellular components involved in the trafficking of proteins from

P. falciparum into an infected erythrocyte. A diagrammatic representation

of the structure of an erythrocyte infected by the parasite P. falciparum.

Some proteins originating from the parasite enter the secretory pathway

via the endoplasmic reticulum (ER), and possibly the Golgi apparatus,

before being shuttled into the erythrocyte across the parasitophorous

vacuole (PV). The PV extends into the interconnected tubulovesicular

network (TVN). The membrane network of the parasite also gives rise to

the Maurer’s clefts, which are thought to play a key role in the export of

proteins of parasitic origin to the erythrocyte. Several molecular chaper-

ones (among them Hsp70s) of parasitic origin are thought to reside in the

PV and Maurer’s clefts, where they are implicated in the trafficking of

proteins from the parasite to the erythrocyte (Vincensini et al. 2005; Lanzer

et al. 2006; Nyalwidhe and Lingelbach 2006). A localized protein transport

network exists inside the parasite and involves the export of proteins from

the cytosol into the mitochondria (Mt) and apicoplast (AP), as represented

by the arrows leading to these organelles. The apicoplast has its own

genome that encodes functions mainly for the maintenance of the

organelle. Parasite nuclear-encoded proteins key to the survival of the

parasite are imported into the apicoplast from the cytosol of the parasite

(Foth et al. 2003). The successful trafficking of these proteins across the

apicoplast membrane very likely requires Hsp70 proteins of the parasite

cytosol and apicoplast.

Shonhai et al.

1804 Protein Science, vol. 16



Furthermore, the fact that chaperones of parasitic originhave been detected in other cell compartments outside theparasite cytoplasm such as the Maurer’s clefts (Fig. 1), isfurther evidence for their possible involvement in theexport of parasite proteins into the erythrocyte (Vincensiniet al. 2005; Lanzer et al. 2006). However, it should be notedthat it seems that the parasite exploits host cell chaperonesduring the assembly of multiprotein subunits at the eryth-rocyte surface (Banumathy et al. 2002). Host Hsp70 andHsp90 in parasite-infected erythrocytes were membrane-associated, whereas the same chaperones were found tooccur in the cytosol of uninfected cells (Banumathy et al.2002). This suggests that in infected cells, host chaperonescould play a role in the trafficking of parasitic proteins intothe erythrocyte (Banumathy et al. 2002).

P. falciparum HSP 70 subfamilies

At least six P. falciparum Hsp70 homologs with featuresspanning across the cytosolic, endoplasmic reticulum(ER), and the mitochondrial forms have been identified(Table 1; Peterson et al. 1988; Sargeant et al. 2006). Thecompartmentalization of Hsp70 protein homologs ineukaryotic cells ensures that they are able to efficientlyundertake specialized cellular roles. A phylogeneticanalysis of Hsp70 proteins from P. falciparum (Fig. 2)illustrates the major Hsp70-like proteins to which theseproteins belong. The cytosolic form of P. falciparumHsp70 (PfHsp70-1) clusters with Hsp70s from eukar-

yotes. PfHsp70-x, a closely related homolog of PfHsp70-1,shows a close phylogenetic link to PfHsp70-1. PfHsp70-2(ER Hsp70 homolog) and PfHsp70-3 (mitochondrial Hsp70homolog) form clusters with their homologs from othereukaryotic organisms. On the other hand, PfHsp70-z andPfHsp70-y (whose molecular mass is at least 100 kDa)form a clade with the Hsp110/Grp170 (Lhs1) subfamily ofHsp70-like proteins. The organization of Hsp70s of para-sitic origin into distinct organelle specific groups withdistinct expression patterns has been documented (Kappeset al. 1993; Olson et al. 1994; Slapeta and Keithly 2004).

Pairwise sequence identity analysis for P. falciparumHsp70s (Table 2) shows a wide range of percentageidentities across these proteins. The highest identity(73%) is between PfHsp70-1 and PfHsp70-x, which isconsistent with phylogenetic data that shows that thetwo proteins are closely related (Fig. 2). PfHsp70-x alsoshares relatively high identity (54%) with the putativeER homolog PfHsp70-2. PfHsp70-2 and PfHsp70-3(mitochondrial homolog) share 46% identity betweenthem. The rest of the proteins share very low identitybetween them, signifying that P. falciparum Hsp70s sharea divergent range of identities between them that mightbe an indication of the wide range of activities that thesechaperones could play across the different organellelocations in which they occur. It has been estimated thatin vitro cultures of P. falciparum produce a minimum of87% of total protein as new proteins in 48 h, and thisfigure rises to at least 90% in a 60-h growth cycle

Table 1. Hsp70s from Plasmodium falciparum

Features

Name of Hsp70 used in this study (bold), alternate name(s), and PlasmoDB annotation

PfHsp70-1a

PfHspe

PfHsp70–1b

PF08_0054

PfHsp70-2a,c

PfGrp78d

PF10875w

PfHsp70-x—

MAL7P1.228

PfHsp70-3a

PfmtHsp70e

PF11_0351

PfHsp70-z—

PF07_0033

PfHsp70-y—

MAL13P1.540

Approximate molecular

mass (kDa) 74 73 76 73 100 108

Chromosome 8 9 7 11 7 13

Cellular location Nucleus and

cytoplasmdERd Cytoplasmf

N.C.

Mitochondrione,f

N.C.

Cytoplasmf

N.C.

ERf

N.C.

Signal sequence/Special

feature

C-terminal EEVD

motif

GGMP motif a

Homolog of

Bip/Grp78d,g

C-terminal ER

sequenced

C-terminal

EEVN motif

Homolog of

mtHsp70e,f

Mitochondrial

transit sequencee,f

Homolog of

Cytoplasmic

Hsp105f

C-terminal

ER sequencef

Expression phase Exoerythrocytic

stageaExoerythrocytic

stageaN.E. N.E. N.E. N.E.

(N.E.) Not yet established; (N.C.) not experimentally confirmed; (ER) endoplasmic reticulum.a Sharma (1992).b Biswas and Sharma (1994).c Nyalwidhe and Lingelbach (2006).d Kumar et al. (1991).e Slapeta and Keithly (2004).f Sargeant et al. (2006).g Kumar and Zheng (1992).

Hsp70s of Plasmodium falciparum

www.proteinscience.org 1805



(Nirmalan et al. 2004). Given the challenges thatP. falciparum encounters in its life cycle, it is not sur-prising that this organism has Hsp70 homologs that havea relative degree of structural diversity in order to be able

to deal with the protein-folding challenges that it man-ages. This is extremely important in the P. falciparum lifecycle since the development of malaria fever adds afurther strain to its protein-folding machinery.

Figure 2. Phylogenetic analysis of P. falciparum Hsp70s. Hsp70-like proteins from P. falciparum (bold) and other sources were

analyzed. NCBI accession numbers of proteins from other organisms, besides P. falciparum, are as follows: Theileria annulata Hsp110

(TaHsp110, XP_952474); Arabidopsis thaliana Hsp110 (AtHsp110, NP_567510); yeast Lhs1 (Lhs1, P36016); Escherichia coli ClpB

(ClpB, AAB49540); human Hsp100 (NP_006651); Agrobacterium tumafaciens DnaK (AgtDnaK, AAR84665); E. coli DnaK

(EcDnaK, BAA01595.1); Trypanosoma cruzi mitochondrial Hsp70 (TcmtHsp70, AAA30215); human BiP (CAA61201); T. annulata

Hsp70 (TaHsp70, A44985); Plasmodium berghei Hsp70 (PbHsp70, AAL34314); T. cruzi Hsp70 (TcHsp70, P05456); and human Hsc70

(AF352832). The Hsp70-like proteins were subdivided into different subgroups as shown on the right-hand side. Dendrograms were

constructed using the BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html)–Protdist neighbor phylogenetic analysis tool. The

TreeView (Page 1996) option housed on the BioEdit program was used to generate the dendrograms.

Table 2. Percentage identities for Hsp70s from Plasmodium falciparum

PfHsp70-1 PfHsp70-2 PfHsp70-3 PfHsp70-x PfHsp70-y PfHsp70-z

PfHsp70-1 — 53 43 73 16 17

PfHsp70-2 53 — 46 54 18 15

PfHsp70-3 43 46 — 46 16 16

PfHsp70-x 73 54 46 — 16 19

PfHsp70-y 16 18 16 16 — 20

PfHsp70-z 17 15 16 19 20 —

Shonhai et al.




Cytosolic and nuclear Hsp70 proteins

Of the six Hsp70s from P. falciparum, it is only PfHsp70-1 (Table 1) that has received widespread research atten-tion with respect to its chaperone properties (Sharma1992; Matambo et al. 2004; Ramya et al. 2006) and as apotential vaccine candidate (Kumar et al. 1990; Behret al. 1992). PfHsp70-1 is a cytosolic/nuclear-localizedP. falciparum Hsp70 (Kappes et al. 1993). The proteinis largely confined to the cytosol, and its nuclear local-ization is enhanced in response to heat stress (Kappeset al. 1993). PfHsp70-1 has a molecular mass of ;74kDa, and possesses the C-terminal EEVD motif, charac-teristic of eukaryotic cytosolic Hsp70s. The EEVD motifof Hsp70 binds to cochaperones including Hop, whoserole is to facilitate the partnership between Hsp70 andHsp90 (Demand et al. 1998). PfHsp70-x, which sharesclose identity to PfHsp70-1, has a molecular mass of ;76kDa (Table 1). However, PfHsp70-x contains a C-terminalEEVN motif in place of the EEVD motif. PfHsp70-xcould be an alternate cytosolic Hsp70 of P. falciparum(Sargeant et al. 2006). There is very little informationon the role of PfHsp70-x, and perhaps it represents theconstitutively expressed cytosolic Hsp70 form of P.falciparum. The fact that both PfHsp70-1 and PfHsp70-xshare high sequence identity, with both possessinghighly conserved bipartite nuclear localization signals(Fig. 3A; Robbins et al. 1991), strongly suggests thatPfHsp70-x, like PfHsp70-1, may also move into thenucleus.

The expression of PfHsp70-1 at the blood stages of theparasite has been confirmed, and this protein has beenreported to be soluble (Joshi et al. 1992; Kappes et al.1993). Although PfHsp70-1 is primarily localized to thecytosol and nucleus (Kappes et al. 1993), it is has beenreported to be present in the parasitophorous vacuole(Nyalwidhe and Lingelbach 2006). It has also beendetected in the Maurer’s clefts (Vincensini et al. 2005),a structure that is thought to be connected to the parasitebut that extends into the erythrocyte (Langreth et al.1978). This has raised the possibility that this proteincould be exported into the erythrocyte. The detection ofantibodies to PfHsp70-1 in malaria patients suggests thatthis protein could join the host circulatory system,thereby invoking an immune response (Kumar et al.1990). Alternatively, PfHsp70-1’s localization in theparasitophorous vacuole and the Maurer’s clefts suggestsa role for this chaperone in the export of P. falciparumproteins into the erythrocyte.

PfHsp70-1 has relatively high basal ATPase activityand ATP-dependent in vitro chaperone activity (Matamboet al. 2004; Ramya et al. 2006). In addition, PfHsp70-1displayed in vivo chaperone function by reversing thethermosensitivity of an Escherichia coli dnaK mutant

strain (Shonhai et al. 2005). The protein also possesses afunctional linker region since mutations in this segmentof the protein resulted in loss of functionality in vivo(Shonhai et al. 2005). Using a chimeric protein composedof the ATPase domain of E. coli DnaK fused to thepeptide-binding domain of PfHsp70-1, evidence for inter-domain communication between the two domains of theseproteins was established (Shonhai et al. 2005). The factthat both full-length PfHsp70-1 and its chimeric deriva-tive composed of the ATPase domain of E. coli DnaKlinked to the peptide-binding domain of PfHsp70-1displayed in vivo function in E. coli DnaK756 cellssuggests that the peptide-binding domain of PfHsp70-1was able to interact with E. coli DnaK substrates (Shonhaiet al. 2005). Furthermore, a recent study showed that theJ-domains (essential for the interaction of Hsp40s withtheir Hsp70 counterparts) of P. falciparum Hsp40 proteins(PfJ4 and PfJ1) were able to interact with E. coli DnaKin vivo (Nicoll et al. 2007). This shows that there is ahigh degree of functional overlap between E. coli andP. falciparum chaperone systems.

Although information on the direct chaperone role ofPfHsp70-1 in P. falciparum is scanty, there is evidencethat points to its possible role in actin polymerization(Tardieux et al.1998). Actin polymerization is a phenom-enon that implicates the role of actin filaments infacilitating host cell invasion by parasites (Dobrowolskiet al. 1997). Furthermore, the fact that PfHsp90 is es-sential for the parasite’s survival places PfHsp70-1 intothe spotlight, since PfHsp70-1 and PfHsp90 have beenobserved to associate in ATP-dependent complexes,suggesting that the two proteins could form a functionalpartnership during the growth and development ofP. falciparum (Banumathy et al. 2003).

A translation initiation factor, 2a (eIF-2a), has beenidentified in P. falciparum whose phosphorylation inhib-its protein synthesis, leading to cell death (Surolia andPadmanaban 1991). Hsp70 has been shown to suppressthe activity of a heme-regulated kinase, thus preventingthe phosphorylation of eIF-2a and indirectly allowingprotein synthesis to proceed (Ramya et al. 2007a). Theantimalarial 15-deoxyspergulain (DSG) has been pro-posed to indirectly inhibit protein synthesis by titratingHsp70 protein and promoting the phosphorylation ofeIF-2a (Ramya et al. 2007a). Ramya et al. (2006) haveprovided evidence that DSG was able to modulate theactivity of PfHsp70-1, possibly by interacting with itsC-terminal EEVD motif. Therefore PfHsp70-1 has beenproposed to be indirectly linked to the regulation ofprotein translation in P. falciparum (Ramya et al.2007b). However, since the EEVD motif is in closeproximity to the peptide-binding domain, by binding ator near the EEVD motif of PfHsp70-1, DSG may also inhibitprotein folding by interfering with substrate binding by





PfHsp70-1. Furthermore, it has been proposed that bysequestering PfHsp70-1, DSG inhibits the trafficking ofsome nuclear-encoded proteins into the apicoplast, killingthe parasite (Ramya et al. 2007b). This is consistent with

the proposed role for P. falciparum Hsp70s in the traffick-ing of proteins destined for the apicoplast (Foth et al. 2003).Proteins that are meant for uptake by the apicoplast areenriched in Hsp70-binding sites, and mutation of these sites

Figure 3. (Continued on next page)


Shonhai et al.



Figure 3. Amino acid sequence alignments of P. falciparum Hsp70s against E. coli DnaK and human Hsc70. (A) Alignment of the amino

acid sequences for the ATPase domains of the P. falciparum Hsp70s against corresponding sequences of E. coli DnaK (BAA01595.1) and

human Hsc70 (AF352832). Some of the key residues in the ATPase domain are highlighted as follows: (purple box) N-terminal ER leader

sequence; (red box) the bipartite nuclear localization signal (Robbins et al. 1991); and (blue boxes with broken lines) highly conserved

residues associated with heat shock proteins. Based on the DnaK sequence, some residues that are important for its function and their

corresponding residues in Hsc70 and PfHsp70 are highlighted as follows: (blue boxes with continuous lines) P143 (proline allosteric

switch) and R151, both of which are important for interdomain function (Vogel et al. 2006a); (green boxes) Y145, N147, D148, N170,

and T173, which interact with DnaJ (Gassler et al. 1998; Suh et al.1998); and (yellow box) T199, a DnaK phosphorylation site (McCarty

and Walker 1991). The different motifs that interact with subunits of the nucleotide (phosphate 1, phosphate 2, and adenosine) are shown.

These motifs are linked by residues represented by the segments connect 1 and connect 2. The numbers on the left-hand side represent the

positions of residues in the respective proteins. (B) An alignment of amino acid sequences of the peptide-binding domains of P.

falciparum Hsp70s, E. coli DnaK, and human Hsc70. Highlighted by the different rectangular boxes are the following residues: (blue box)

linker; (orange box) terminal EEV-motifs; (yellow boxes) the terminal Hsp70 ER retention signal (Pelham 1989); (arrows) residues that

are in contact with the substrate (Zhu et al. 1996), of which those that constitute the hydrophobic arch and hydrophobic pocket (Mayer

et al. 2000) are highlighted by red arrows and a green arrow, respectively. (Blue arrows) The rest of the residues that occur in contact with

substrate; (red boxes) residues that constitute the different subdomains of the b-sheet. (Black line) Residues constituting the different lid

subdomains (A–E) of DnaK; (blue lines) residues constituting the lid subdomains of Hsc70 (Chou et al. 2003). (White on black

background) Identical residues; (black on gray background) similar residues. The BioEdit program ClustalW (http://www.mbio.ncsu.edu/

BioEdit/bioedit.html) (Thompson et al. 1994) based alignment option was used to carry out sequence alignment. The numbers on the left-

hand side represent the positions of residues in the respective proteins.




led to mistargeting of the proteins in P. falciparum cells(Foth et al. 2003).

Heat shock protein 70 interacting protein (Hip) is acochaperone of Hsp70 that competes with Bag-1 for acontact site in the ATPase domain of Hsp70 and acts indirect opposition to Bag-1 by stabilizing the Hsp70–peptide complex (Hohfeld et al. 1995; Hohfeld andJentsch 1997). Hip’s ability to regulate Hsp70 functionin vivo has been confirmed (Nollen et al. 2001). Ramyaet al. (2006) observed that P. falciparum Hip (PFE1370w)was able to modulate the chaperone function of PfHsp70-1and PfHsp70-3 in vitro. Therefore, there is growinginterest in identifying cochaperones that modulate thechaperone activity of PfHsp70-1 and their mechanism ofaction.

Allosteric communication is an essential feature ofHsp70 protein function, regulating both substrate releaseand ATP hydrolysis (Han and Christen 2003). Theelucidation of the partial full-length structure of bovineHsc70 by Jiang et al. (2005) revealed structural detailsthat suggest that the interaction between Hsp70 andHsp40 occurs at the linker interface, allowing Hsp40 tointeract with both the ATPase and peptide-bindingdomain of Hsp70. Not surprisingly, some residues onHsp70 that are important for interaction with Hsp40 areimplicated in interdomain communication (Gassler et al.1998; Jiang et al. 2005). Hsp70 residues implicated ininterdomain communication and their interaction withHsp40 are conserved in PfHsp70-1 (Fig. 3A,B; Gassleret al. 1998; Jiang et al. 2005; Vogel et al. 2006b). The factthat it has previously been shown that PfHsp70-1 pos-sesses a functional linker whose disruption compromisedits function, suggests that the linker region of PfHsp70-1is an important structural component of its function(Shonhai et al. 2005). Except for PfHsp70-y andPfHsp70-z, the rest of the P. falciparum Hsp70s displayhighly conserved linker regions (Fig. 3B), suggesting thatthis motif might be important in the modulation of theirfunction.

Endoplasmic reticulum Hsp70

PfHsp70-2 is a homolog of the mammalian ER 78-kDaglucose-regulated protein (Grp78), also referred to asimmunoglobulin-binding protein (BiP) (Kumar andZheng 1992; Kappes et al. 1993). The protein has amolecular mass of ;73 kDa (Table 1; Kumar and Zheng1992). PfHsp70-2 possesses the ER N-terminal leadersequence and a possible C-terminal ER retention signal(SDEL) that differs from that of Grp78/BiP, whichcontains a typical eukaryotic C-terminal ER retentionsignal (KDEL) (Pelham 1989). Indeed, PfHsp70-2 hasbeen observed to be largely confined to ER-like struc-tures in P. falciparum (Kumar et al. 1991). In addition,

PfHsp70-2 has been detected in the Maurer’s clefts(Vincensini et al. 2005). Although PfHsp70-2 wasslightly induced in response to heat stress, no inductionof the protein was invoked by partial glucose deprivationand other known inducers of Grp78 proteins (Lee 1987;Kumar et al. 1991).

PfHsp70-2 shows a very close phylogenetic link withhuman BiP (Fig. 2). Although there is no study that hasbeen conducted to verify the direct chaperone role ofPfHsp70-2 in P. falciparum, a study by LaCount et al.(2005) based on the yeast two-hybrid system (Fig. 4)showed that PfHsp70-2 potentially interacts with moreP. falciparum proteins than PfHsp70-1 and PfHsp70-3(the mitochondrial Hsp70 homolog). This study impli-cated PfHsp70-2 in association with the following proteinfamilies: cytoskeletal and membrane proteins, transla-tional and transcriptional machinery, proteosome andproteolytic enzymes, enzymes involved in physiologicaland metabolic pathways, and DNA repair and replicationenzymes. Human BiP is known to interact with proteinsexported to the ER, maintaining them in states ideal eitherfor export or degradation (Gething 1999). Assuming thatPfHsp70-2 also serves the same purpose, it could explainthe possible association of PfHsp70-2 with a widespectrum of P. falciparum proteins (Fig. 4; LaCountet al. 2005). One of the proteins that potentially interactswith PfHsp70-2 is the erythrocyte membrane protein(PFI1830c) (Fig. 4), a protein that is exported into theerythrocyte (Baruch et al. 1996). Therefore, the potentiallocalization of PfHsp70-2 to the Maurer’s clefts (Vincensiniet al. 2005) could be perceived as a strategy to promote theexport of P. falciparum proteins into the erythrocyte.

Mitochondrial Hsp70

PfHsp70-3 is the mitochondrial Hsp70 homolog (Slapetaand Keithly 2004; Sargeant et al. 2006). The mitochon-drial and cytosolic Hsp70 homologs from the apicom-plexan kingdom and other closely related species displaydistinct phylogenetic features, suggesting that these pro-teins have distinct roles (Slapeta and Keithly 2004).Despite the fact that this protein possesses a weakly con-served mitochondrial pre-sequence, it is phylogeneticallyrelated to its mitochondrial Hsp70 from other eukaryoticorganisms (Slapeta and Keithly 2004). PfHsp70–3 formsa monophyletic clade with its Trypanosoma cruzi mito-chondrial Hsp70 homolog (TcmtHsp70), which clustersclosely with the prokaryotic cytosolic Hsp70s fromEscherichia coli (EcDnaK) and Agrobacterium tumafa-ciens DnaK (AgtDnaK) (Fig. 2).

There is very little experimental information on thepossible role of PfHsp70-3. In eukaryotic cells, mito-chondrial Hsp70 proteins play an important role in theimport of pre-proteins into the mitochondria (Bauer et al.

Shonhai et al.




2000). However, it is yet to be established whetherPfHsp70-3 is involved in the import of P. falciparumpre-proteins from the parasite cytosol to the mitochon-drion. However, the high homology between PfHsp70-3and human HspA9B has been presented as evidence thatPfHsp70-3, like HspA9B, could be involved in the exportof proteins since HspA9B is implicated in antigenprocessing and protein export (Wadhwa et al. 2002; Bothaet al. 2007).

A study based on the yeast two-hybrid system (Fig. 4)shows that some of the proteins associated with PfHsp70-3are a malaria antigen (MAL13P.304) and two asparagine-rich antigen proteins (PF08_0060 and PF11_0111). Thissuggests a possible role of PfHsp70-3 during proteintrafficking into the erythrocyte since both malaria antigenand asparagine-rich proteins are exported into the eryth-rocyte (Weber et al. 1988; Barale et al. 1997). Perhapsthe fact that PfHsp70-3 has been detected in the Maurer’s

Figure 4. The potential P. falciparum Hsp70 interactome of proteins. The network of interactions involving Hsp70s from

P. falciparum and other proteins from the parasite was constructed based on data from yeast two-hybrid studies (LaCount et al.

2005). Chaperones: (solid circle) Hsp70s; (solid square) Hsp40-like proteins; (solid arrow) PfHip; (open rectangle) disulfide isomerase.

The rest of the proteins involved in the network are classified into the following subgroups: (open oval) cytoskeletal and membrane

proteins; (open rectangle) translational and transcriptional machinery; (open rhombus) proteosome and proteolytic enzymes; (dotted

rectangle) enzymes involved in physiological and metabolic pathways; and (open star) DNA repair and replication enzymes. The arrow

points in the direction of the prey. Reciprocal association is represented by arrows facing opposite directions. A broken arrow links

PfHsp70-3 and PFL1385c. Self-association of chaperones is illustrated by two adjacent shapes representing the particular protein. The

interactors are represented by their locus numbers.





clefts (Vincensini et al. 2005; Lanzer et al. 2006) suggeststhat this chaperone might facilitate the export of proteins ofparasitic origin into the erythrocyte. Assuming thatPfHsp70-3 occurs in several cellular organelles as is truefor HspA9B, perhaps it is not surprising that PfHsp70-3could be found in the parasite mitochondrion and Maurer’sclefts in the infected erythrocytes. However, it is confound-ing to reconcile how the same protein could act as a proteinimport system (in the mitochondrion) and as a possibleprotein export system (in the Maurer’s clefts). Therefore,the proposed localization of PfHsp70-3 as observed byVincensini et al. (2005) may need to be verified byindependent localization experiments.

Hsp110/Grp170 homologs

PfHsp70-y and PfHsp70-z have approximate molecularmasses of 100 kDa and 108 kDa, respectively. Althoughthe two Hsp70-like proteins have relatively conservedATPase domains, they display very low conservation inthe peptide-binding domains (Figs. 3A,B). Of the sixP. falciparum Hsp70s, it is only these two that do not havea conserved nuclear localization signal (Robbins et al.1991). PfHsp70-z and PfHsp70-y also do not have thethreonine residue corresponding to the T199 of DnaK,which is a crucial phosphorylation site of DnaK (McCartyand Walker 1991). Generally, the linker is conserved inall the Hsp70s from P. falciparum, except in PfHsp70-yand PfHsp70-z. In addition, the fact that both PfHsp70-yand PfHsp70-z lack a distinct linker structure that iscrucial for allosteric control of Hsp70s (Han and Christen2001; Vogel et al. 2006b) suggests that these Hsp70s areregulated differently or have a distinct role that isdivergent from the typical Hsp70 chaperone function.The close phylogenetic association of PfHsp70-y andPfHsp70-z with Hsp70-like proteins of higher molecularmasses such as TaHsp110 from another apicomplexanspecies, Theileria annulata, and AtHsp110 from a plantspecies (Arabidopsis thaliana) hints at the fact that thetwo Hsp70-like proteins are Grp110/Grp170 homologs.The Grp110/Grp170 family of proteins has structural–functional features distinct from Hsp70s, although theyalso have some degree of similarity to Hsp70s (Eastonet al. 2000). The Hsp110/Grp170 family of proteins has alonger helical lid compared to Hsp70 proteins, whichenables them to bind larger amounts of substrate (Eastonet al. 2000). The fact that PfHsp70-y contains a putativeterminal ER-retention signal and shows close phyloge-netic connection to the yeast Hsp70-like Hsp110/Grp170homolog Lhs1 (Steel et al. 2004) hints at the possibilitythat PfHsp70-y could be a NEF for PfHsp70-2. BesidesLhs1 (Steel et al. 2004), another yeast Hsp110, Sse1p hasbeen identified as a NEF (Dragovic et al. 2006). There-fore, PfHsp70-y possibly acts as the ER-based NEF of the

Hsp110/Grp170 family, while PfHsp70-z is the cytosolicHsp110/Grp170 protein of P. falciparum.

Characterization of the chaperone featuresof P. falciparum Hsp70s

Although relatively divergent in structure, P. falciparumHsp70s must display conserved features that are importantfor their chaperone role. These include key residues forinteraction with their Hsp40 partners, NEFs, and peptidesubstrates. Since PfHsp70-1 is the most extensively studiedP. falciparum Hsp70 protein, its key functional residueswere analyzed relative to the structures of the well-studiedE. coli DnaK and human Hsc70. Since there is evidence thatPfHsp70-1 could interact with a subpopulation of E. colisubstrates in vivo (Shonhai et al. 2005), we analyzed itsstructural features important for chaperone activity com-pared to E. coli DnaK and human Hsc70, with particularreference to residues that interact with cochaperones andsubstrate. The structural comparison between PfHsp70-1and Hsc70 was used in order to identify important structuralmotifs that are conserved between the two Hsp70s ofeukaryotic origin. Furthermore, a comparative analysis ofthe chaperone properties of PfHsp70-1 and human Hsc70could be used to establish whether the two proteins could beregulated by the same cochaperones since it has beenhypothesized that human Hsp70s could be regulated byP. falciparum Hsp40s that are exported into the erythrocyte(Sargeant et al. 2006; Botha et al. 2007).

P. falciparum Hsp70 residues important for interactionwith substrate

Only PfHsp70-1 and PfHsp70-x possess the typicaleukaryotic Hsp70 arch residues that are inverted com-pared to those of E. coli DnaK (the DnaK arch is made upof M404 and A429, and most eukaryotic Hsp70s have Aand Y at corresponding positions) (Fig. 3B; Zhu et al.1996). PfHsp70-2 has residues V/Y, while PfHsp70-3 hasresidues L/A at the arch position. However, bothPfHsp70-2 and PfHsp70-3 have the conserved valineresidue in the hydrophobic pocket. PfHsp70-z andPfHsp70-y have arch residues different from those ofPfHsp70-1 and PfHsp70-x, and neither of the two possessthe conserved valine residue associated with the hydro-phobic pocket position. Instead, PfHsp70-z and PfHsp70-yhave leucine and isoleucine residues, respectively, in theposition corresponding to the Hsp70 hydrophobic pocket.The lack of consensus in arch residues observed betweencytosolic P. falciparum Hsp70s and the rest of theirhomologs is consistent with the fact that arch residues arethe least-conserved substrate-binding residues, thus per-fectly placing them as determinants of Hsp70 substratespecificity (Rudiger et al. 1997; Mayer et al. 2000). Based

Shonhai et al.




on sequence alignment data, PfHsp70-1, like other Hsp70s(Fig. 3B; Zhu et al. 1996), possesses a substrate-bindingcavity that is rich in hydrophobic residues.

Phosphorylation of Hsp70 proteins in P. falciparum

Phosphorylation of Hsp70 proteins is an important func-tional regulatory aspect of their chaperone role (McCartyand Walker 1991). The concomitant expression andphosphorylation of major chaperones from Plasmodiumhas been observed (Wiser and Plitt 1987; Kappes et al.1993). PfHsp70-1 and PfHsp70-2 display growth-phase-dependent phosphorylation in vivo through their threo-nine and serine residues (Kumar et al. 1991; Kappeset al. 1993). The phosphorylation of Hsp70 proteins isimportant in the regulation of their activities (McCartyand Walker 1991; Cvoro et al. 1999). The concomitantexpression and phosphorylation of Hsp70 proteinsensures that their chaperone activity is enhanced duringstressful conditions (McCarty and Walker 1991). There-fore, the fact that at least four of the P. falciparum Hsp70spossess a threonine residue that corresponds to the DnaKphosphorylation site (T199) (Figure 3A; McCarty andWalker 1991) suggests that most of them may havephosphorylation-regulated activities.

Using the KinasePhos phosphorylation prediction tool(Huang et al. 2005a,b), potential phosphorylation sitesin the six Hsp70s from P. falciparum were identified(Fig. 5). Based on this phosphorylation prediction tool,P. falciparum Hsp70s are predicted to be phosphorylatedmostly through serine and threonine residues, in agreementwith a previous observation based on studies on the phos-phorylation of PfHsp70-1 and PfHsp70-2 (Kappes et al.1993). PfHsp70-1 has been observed to be phosphorylatedmore through its serine residues than through threoninesites, while PfHsp70-2 was phosphorylated more throughits threonine than through serine residues (Kappes et al.1993). Based on the predicted number of phosphorylationsites, PfHsp70-1 and PfHsp70-2 each have approximatelythe same number of threonine and serine phosphorylationsites (Fig. 5). Therefore it is not clear why phosphorylationthrough the threonine and serine sites occurs preferentiallythrough one of the two residues in each protein (Kappeset al. 1993). Perhaps this represents a unique regulatorymechanism for the two proteins. However, the fact that nophosphorylation was observed to occur through tyrosineresidues for both PfHsp70-1 and PfHsp70-2 could bebecause of the low number of possible tyrosine phosphor-ylation sites in these two chaperones (Fig. 5; Kappes et al.1993). Compared to their P. falciparum homologs,PfHsp70-y and PfHsp70-z exhibit high numbers of poten-tial tyrosine phosphorylation sites. This is further evidencethat the chaperone properties of these two proteins could bedistinct from the rest of the P. falciparum Hsp70s. This is

particularly the case for PfHsp70-z, which has only onepredicted threonine phosphorylation site.

Regulation of P. falciparum Hsp70sby Hsp40 cochaperones

Several Hsp40-binding sites corresponding to those iden-tified to be important for this process in E. coli DnaK(Gassler et al. 1998; Suh et al. 1999) are generallyconserved in the ATPase domains of P. falciparumHsp70s (Fig. 3A). Watanabe (1997) established thatcertain P. falciparum Hsp40s may be induced by heatstress. At least some of the P. falciparum Hsp70s wouldbe expected to have chaperone activities that are modu-lated by Hsp40s (Botha et al. 2007). At least 43 Hsp40-like proteins are encoded on the P. falciparum genome(Sargeant et al. 2006; Botha et al. 2007). Of these, 19 arepredicted to be exported based on export signal motifsthat they carry (Sargeant et al. 2006; Botha et al. 2007).Therefore, there are 24 Hsp40 proteins that could berestricted to the cytoplasm of P. falciparum. Ironically, allthe P. falciparum Hsp70s do not possess export signalmotifs (Marti et al. 2004; Sargeant et al. 2006) and aretherefore predicted not to be exported into the erythrocyte(Botha et al. 2007). It would be expected that part of thislarge entourage of Hsp40s that P. falciparum possiblyexports into the erythrocyte could serve as cochaperonepartners of host Hsp70s (Sargeant et al. 2006; Botha et al.

Figure 5. Comparative potential phosphorylation status of P. falciparum

Hsp70s. A bar graph representation for the potential phosphorylation status

of the Hsp70 proteins from P. falciparum. The KinasePhos phosphoryla-

tion prediction tool (Huang et al. 2005a,b) was used to predict potential

serine, threonine, and tyrosine residues that could be phosphorylated by

kinases. The bar graphs are for (1) PfHsp70-1, (x) PfHsp70-x, (2) PfHsp70-2,

(3) PfHsp70-3, (y) PfHsp70-y, and (z) PfHsp70-z.





2007). A study showed that host chaperones (includinghuman Hsp70 and Hsp90) interact with P. falciparumproteins exported into the erythrocyte (Banumathy et al.2002). Therefore, these exported P. falciparum Hsp40spossibly scavenge for P. falciparum proteins, directingthem to the host Hsp70 machinery.

It has been proposed that of the 24 Hsp40-like proteinsconfined to the P. falciparum cytoplasm, two type IHsp40s (PfJ1 and PF14_0359) (Watanabe 1997; Sargeantet al. 2006) or a type II Hsp40 (PFB059w) possibly actas cochaperones of PfHsp70-1 (Botha et al. 2007). Itis thought that protein disulfide isomerase (PDI) relatedP. falciparum Hsp40s (PfJ2 and PF13_0102) could serveas the ER-based cochaperones of PfHsp70-2 (Botha et al.2007). Yeast two-hybrid data for PfHsp70-3 suggested ithas possible direct and indirect interactions with a ring-infected erythrocyte surface antigen (RESA)-like protein,and mature parasite-infected erythrocyte surface antigen(MESA) Hsp40-like protein (Fig. 4). These associationscould be partner protein interactions; however, this isdifficult to reconcile with the erythrocyte membranelocalization of the RESA-like and MESA proteins. There-fore, one cannot exclude the possibility that the RESA-like and MESA proteins are substrates of PfHsp70-3during their export to the host.

The possible regulation of P. falciparum Hsp70sby nucleotide exchange factors

There is evidence that Hsp70s differ with respect to theirintrinsic rate of ADP release, justifying the need for themto have specialized nucleotide exchange systems (Russellet al. 1998; Silberg and Vickery 2000). Variations withinstructural features of the NEF-binding site of Hsp70sare responsible for providing unique Hsp70–nucleotideexchange partnerships (Brehmer et al. 2001). For thisreason, Brehmer et al. (2001) subdivided nucleotideexchange systems in Hsp70s into three prototypes: DnaKproteins, HscA (Hsc66), and Hsc70 proteins, as deter-mined by their distinct interaction with GrpE and Bag-1.

Although structurally unrelated to GrpE, Bag-1 isconsidered the functional equivalent of GrpE (Hohfeldand Jentsch 1997). This is because structures of GrpE–DnaK and Bag-1–Hsc70 complexes displayed identicalconformational changes in the ATPase domains of therespective Hsp70 protein (Harrison et al. 1997; Sondermannet al. 2001). On the other hand, the mechanism by whichnucleotide exchange occurs on eukaryotic Hsp70 throughthe action of HspBP1 is distinct from the GrpE/Bag-1-driven one (Shomura et al. 2005). However, despite thefact that GrpE and Bag-1 manifest similar conformationalchanges in Hsp70 proteins, the specific mechanisticdetails in which these two NEFs interact with their Hsp70partners are not the same. GrpE functions as a dimer

(Harrison et al. 1997), as opposed to Bag-1, which uses asingle domain (Sondermann et al. 2001). GrpE has morecontact points with DnaK than exist between Bag-1 andHsc70 (Sondermann et al. 2001). It is becoming clearthat there are variations in the way GrpE and Bag-1operate to effect nucleotide exchange. For example,whereas GrpE triggers the release of both ADP and ATPfrom DnaK, Bag-1 induces only ADP release (Brehmeret al. 2001). HspBP1, though a eukaryotic NEF, bindsand induces nucleotide exchange conformation in Hsp70proteins in a different mechanism from Bag-1 (Shomuraet al. 2005).

The possible role of NEFs in the regulation of P.falciparum Hsp70s was investigated by exploring theconservation of residues crucial for nucleotide exchangein PfHsp70-1. Amino acid sequence alignments wereconducted in order to identify residues known to becrucial for the interaction of Hsp70s with GrpE, Bag-1,and HspBP1. A conserved loop region that occurs insubdomain IB of DnaK is thought to ensure the stableinteraction of DnaK with GrpE (Fig. 6A,B; Buchbergeret al. 1994). This loop is also implicated in the regulationof nucleotide binding by DnaK and its role is essential forDnaK function (Buchberger et al. 1994). In addition, asalt bridge that is formed by R34 and E369 in DnaK thatis conserved in other Hsp70s regulates nucleotide ex-change by preventing nucleotide release in the absenceof GrpE, and opens up to release nucleotide when GrpEbinds to the neighboring loop (Fig. 6A,B; Buchbergeret al. 1994). Salt bridges that are equivalent to the R34–E369 salt bridge of DnaK (Buchberger et al. 1994) arerepresented in both Hsc70 and PfHsp70-1 (Fig. 6A,B). Inaddition, DnaK has two extra salt bridges that linksubdomains IB and IIB that further regulate nucleotideexchange (Brehmer et al. 2001). On the other hand, bothPfHsp70-1 and Hsc70 only have one such salt bridge each(Fig. 6A,B).

Hsp70s are classified as DnaK, HscA, or Hsc70 proteinfamilies according to their mechanism of interaction withNEFs (Brehmer et al. 2001). Key to this classificationsystem are the salt bridges between subdomains IB andIIB and a segment that is referred to as the GrpE signatureloop (Fig. 6A,B; Brehmer et al. 2001). Within the GrpEsignature motif is a substructure, the GrpE motif (Fig. 6A;Brehmer et al. 2001). The GrpE motif in DnaK forms aloop that is essential for its interaction with GrpE (Fig.6A,B; Brehmer et al. 2001). Both PfHsp70-1 and Hsc70have a GrpE motif that is structurally divergent from thatof DnaK (Fig. 6A,B). The fact that PfHsp70-1 has highsequence similarity to human Hsc70 in the critical GrpEmotif section (Fig. 6A–D; Brehmer et al. 2001) suggeststhat NEFs that interact with Hsc70 could also recognizePfHsp70-1. Based on this decisive feature, there is evidencethat PfHsp70-1 falls into the category of cytosolic Hsp70s

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(Fig. 6A,B; Brehmer et al. 2001; Sondermann et al. 2001).Structurally, PfHsp70-1 has salt bridges and motifs impli-cated in nucleotide exchange that are more identical tohuman Hsc70 than E. coli DnaK (Fig. 6A,B).

Of the five Hsc70 residues that interact with bothBag-1 and HspBP1, PfHsp70-1 has four of these residuesidentical to corresponding residues in Hsc70 (Fig. 6C,D).It should be noted that most of the residues implicated

Figure 6. Identification of residues of PfHsp70-1 predicted to be important in nucleotide exchange. (A) Conservation of residues

constituting interdomain loops and salt bridges in DnaK, Hsc70, and PfHsp70 and (B) three-dimensional images for the ATPase domains

of DnaK, Hsc70, and PfHsp70 showing the loops and salt bridges crucial in nucleotide exchange. Residues in subdomain IB (red) form a

highly conserved loop and salt bridge (green) that link subdomains IA and IB. The loop and salt bridge are both important for nucleotide

exchange in DnaK (Buchberger et al. 1994). DnaK has two additional salt bridges that link subdomains IB and IIB (green). Hsc70 and

PfHsp70 each have only one of these. An additional feature that confers specificity to the interaction of NEF with a particular Hsp70 is the

GrpE signature loop, whose (yellow) tip (motif) is made up of highly divergent residues in Hsp70s (Brehmer et al. 2001). The predicted

structures for the ATPase domains of human Hsc70 and PfHsp70 were generated by homology modeling (SWISS-MODEL, first approach

mode) (Schwede et al. 2003) using the structure of bovine Hsc70 as a template (1YUW.pdb) (Jiang et al. 2005). The images of E. coli

DnaK (1DKG.pdb), human Hsc70, and PfHsp70 were rendered using PyMOL (DeLano 2002). The figure was based on a previous

analysis by Brehmer et al. (2001). (C) Residues of Hsc70 that interact with HspBP1 and Bag-1 (Sondermann et al. 2001; Shomura et al.

2005) together with their corresponding residues in PfHsp70 and DnaK are shown. Bag-1 contact sites are red; HspBP1 contact sites (blue)

are highlighted; residues that are implicated as both Bag-1 and HspBP1 contact sites are shown in yellow background. (D) A top view of

the three-dimensional structure of PfHsp70 ATPase domain showing the potential solvent exposure of residues that may have contact with

nucleotide exchange factors: Bag-1 (red); HspBP1 (blue); and contact residues common to both NEFs (yellow). The predicted structure for

the ATPase domain PfHsp70 was generated by homology modeling (SWISS-MODEL, first approach mode; Schwede et al. 2003) using

the structure of bovine Hsc70 as a template (1YUW.pdb; Jiang et al. 2005). The image was rendered using PyMOL (DeLano 2002).





in Bag-1 and HspBP1 contact (Sondermann et al. 2001;Shomura et al. 2005) are not only conserved in PfHsp70-1,but they are also predicted to be solvent-exposed (Fig. 6D),suggesting their accessibility for protein–protein interac-tion (Rajamani et al. 2004). It is therefore conceivable thatnucleotide exchange in PfHsp70-1 could occur throughthe same mechanism as that of Hsc70, since most of theresidues of PfHsp70-1 implicated in its interaction withNEF are similar to those of Hsc70. P. falciparum containsa GrpE homolog (PF11_0258). However, GrpE homologsof eukaryotic origin such as yeast are located in themitochondria (Westermann et al. 1995). Therefore, sincePfHsp70-1 is cytosolic, PfGrpE may not be its NEF.

Conclusion and future perspectives

P. falciparum displays a functionally and structurallydiverse group of Hsp70 proteins. Although we have verylittle information on the role of these proteins, studies thathave been conducted so far strongly suggest a pivotal rolefor Hsp70 proteins in the life cycle and pathogenicity ofP. falciparum. It is clear that Hsp70 proteins play animportant role in the translation and export of proteinsinto the apicoplast (Foth et al. 2003; Ramya et al. 2007b).Hsp70 proteins could be important for sustaining reac-tions in the apicoplast of the parasite. This finding begsfor further research attention to establish the direct inputof P. falciparum Hsp70s in this process. This is importantsince the apicoplast hosts metabolic pathways that aredistinct from those of the host cell, making it a target fordrug design (Ralph et al. 2001). Furthermore, cochaper-ones that are responsible for the modulation of thechaperone activities of the different Hsp70s in P. falci-parum need to be identified. For example, the identifica-tion of cochaperones of PfHsp70-1 is long overdue sincethe role of this protein in the life cycle of the parasite hasbeen highlighted. Perhaps most interesting is the possibledevelopment of drugs that target Hsp70 function in P.falciparum. However, because of the high conservationbetween human Hsp70s and P. falciparum Hsp70s, thedesign of potential antimalarials targeting P. falciparumHsp70s is a real challenge. A more feasible approachmight be the design of drugs that interfere with the Hsp70interactome in P. falciparum. An example of this will beto identify inhibitors that may selectively interfere withthe possible partnership between PfHsp70-1 andPfHsp90, without interfering with the functional partner-ship between host cell Hsp70 and Hsp90 proteins.

Acknowledgments

This work was funded in part by a Wellcome Trust Grant(066705; United Kingdom), a National Research FoundationGrant (NRF; South Africa), and a Medical Research Council

Grant (MRC; South Africa), all awarded to G.L.B. A.S. wasawarded an NRF Ph.D. Grant-Holder Bursary and a Ph.D.Scholarship from the Cannon Collins Educational Trust ofSouthern Africa.

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The structural and functional diversity of Hsp70 proteins ... · Since chaperones constitute one of the major protein families in the parasitophorous vacuole in an infected erythrocyte