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.

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.

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).

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 chaperones (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.

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 (Gässler 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 β-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.

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 phosphorylation 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.