A systematic classification of Plasmodium falciparum P-loop NTPases: structural and functional correlation
- Deepti Gangwar†1,
- Mridul K Kalita†2,
- Dinesh Gupta2,
- Virander S Chauhan1 and
- Asif Mohmmed1Email author
© Gangwar et al; licensee BioMed Central Ltd. 2009
Received: 29 December 2008
Accepted: 18 April 2009
Published: 18 April 2009
The P-loop NTPases constitute one of the largest groups of globular protein domains that play highly diverse functional roles in most of the organisms. Even with the availability of nearly 300 different Hidden Markov Models representing the P-loop NTPase superfamily, not many P-loop NTPases are known in Plasmodium falciparum. A number of characteristic attributes of the genome have resulted into the lack of knowledge about this functionally diverse, but important class of proteins.
In the study, protein sequences with characteristic motifs of NTPase domain (Walker A and Walker B) are computationally extracted from the P. falciparum database. A detailed secondary structure analysis, functional classification, phylogenetic and orthology studies of the NTPase domain of repertoire of 97 P. falciparum P-loop NTPases is carried out.
Based upon distinct sequence features and secondary structure profile of the P-loop domain of obtained sequences, a cladistic classification is also conceded: nucleotide kinases and GTPases, ABC and SMC family, SF1/2 helicases, AAA+ and AAA protein families. Attempts are made to identify any ortholog(s) for each of these proteins in other Plasmodium sp. as well as its vertebrate host, Homo sapiens. A number of P. falciparum P-loop NTPases that have no homologue in the host, as well as those annotated as hypothetical proteins and lack any characteristic functional domain are identified.
The study suggests a strong correlation between sequence and secondary structure profile of P-loop domains and functional roles of these proteins and thus provides an opportunity to speculate the role of many hypothetical proteins. The study provides a methodical framework for the characterization of biologically diverse NTPases in the P. falciparum genome.
The efforts made in the analysis are first of its kind; and the results augment to explore the functional role of many of these proteins from the parasite that could provide leads to identify novel drug targets against malaria.
Despite encouraging advances in vaccine development, malaria remains the most serious and widespread parasitic disease of humans. Each year, approximately 300–500 million people become infected with malaria and two to three million die as a result . The availability of the complete genome sequence of Plasmodium falciparum, the causative agent of fatal cerebral malaria, has opened new avenues to identify genes important for the parasite's survival. This information can be utilized for the development of effective drugs or vaccines against the parasite. Unfortunately, nearly 60% of the P. falciparum genome (5411 proteins) has been designated as hypothetical proteins as they lack sequence similarity to any protein known to date . This large and unexplored group of hypothetical proteins may contain proteins that play an important role in physiological pathways specific to the malaria parasite. The functional interruption of such proteins/pathways without deleterious consequences to the host should be the one of the primary tasks in data mining. A pipeline of systematic studies is thus required to elucidate the functional relevance of such proteins in the parasite's survival.
A number of these hypothetical proteins (unknown protein function) in P. falciparum genome contain a P-loop NTPase domain. Briefly, the P-loop NTPases constitute a large super-family of proteins  and are involved in disparate physiological processes. For example, processes include translation, transcription, replication and DNA repair, intracellular trafficking, membrane transport and activation of various metabolites [3–5]. The P-loop NTPases carry out such diverse cellular functions by hydrolyzing the α-β phosphate bond of a bound nucleotide triphosphate i.e. ATP or GTP .
Based on amino acid sequence, the P-loop NTPase fold is characterized by the presence of a N-terminal Walker A motif, represented by a flexible loop joining a β-strand and an α-helix. The loop typically adopts the sequence pattern GxxGxGK [ST], whose function is to properly position the triphosphate moiety of a bound nucleotide. The distal Walker B motif (hhhhDE) contains a conserved aspartate (less commonly glutamate residue). The motif is situated terminally in a β-strand and binds a water-bridged Mg2+ ion [5, 7]. On structural aspects, the P-loop NTPases are α-β proteins that contain regularly recurring α-β units with the five β-strands (β1–β5). The β-strands forms a central core arranged in the order β(5-1-4-3-2) or β(5-1-3-4-2), surrounded by α-helices on both sides [8, 9]. The P-loop NTPases can be divided into two groups: one group includes the nucleotide kinases and the GTPases where the β-strand leading to the P-loop and the Walker B strand are direct neighbors. The second group includes AAA, ABC, SF1/2 helicases and RecA/F1 ATPases and is characterized by an additional β-strand inserted between the P-loop strand and the Walker B strand [10–12]. Despite these basic common sequence and structural features, the P-loop NTPases exhibit extreme sequence divergence. The huge sequence diversity had so far hampered a clear understanding of the phylogenetic relationships within these P-loop proteins. The entire complement of the P-loop NTPases of an organism varies considerably in sequence and the functional aspects. Therefore, identification and phylogenetic classification of the P-loop NTPases of an organism may provide insights into distinct physiological processes involving these proteins. Since the malaria parasite resides inside the host cells during most part of its life cycle, some of these processes such as trafficking of proteins and translocation of metabolites might be unique to the parasite and involve P-loop NTPases. In the present study, a comprehensive survey of the P-loop NTPases in P. falciparum is carried out by analyzing the sequence and structural features of the NTPase domain of functionally diverse proteins. Further, based on these identified features, a few of these hypothetical proteins are classified. For robustness of the analysis, the traditional cladistic and phylogenetic tree approaches (based on sequence and structural motifs of P-loop NTPases) are also combined. The study underlines the evolutionary information in addition to other sequence-structure features and thus, to develop a systematic classification of the P-loop NTPases in the P. falciparum. The study has also facilitated identification of some of the parasite specific P loop-NTPases as putative novel drug targets against the parasite.
Retrieval of sequences with classical Walker A and Walker B motifs
An initial computational search of the complete P. falciparum genome for the Superfamily: P-loop NTPase (SSF52540) revealed 302 proteins; 67 out of which are annotated as hypothetical proteins. In addition, to extract the proteins with classical Walker A and Walker B motifs and their variants, the motif search tool of PlasmoDB release 4.4 http://v4-4.plasmodb.org/restricted/plasmodbmotif.shtml is used. These two motifs are represented by GxxGxGK [TS] and hhhhDE patterns, respectively where 'x' is any of the 20 amino acids and 'h' is any hydrophobic residue. It led to the inclusion of those proteins which have been annotated as P-loop NTPases by PlasmoDB and Pfam databases. From this analysis, an initial set of 120 P-loop NTPase sequences containing both Walker motifs is obtained. Further, for each of these proteins; the sequence regions corresponding to the P-loop domain only (comprising both Walker regions) are extracted. Variation in the sequence length (171 to 8094 amino acids) and Walker A and Walker B motifs is observed for most of these proteins. The unavailability of crystal structures of most of these proteins is a major hindrance in the structure based multiple alignments and their systematic analysis; hence only 97 proteins in which the P-loop domain encompasses a length of 300 to 400 residues are studied, to facilitate in sequence based multiple alignments (Additional file 1). The refined dataset is then used for secondary structure and phylogenetic analyses, followed by classification and orthology studies. Apart from above analysis, the protein sequences are also screened using signalP 3.0, TMHMM v2.0 and TMpred tools to predict signal and transmembrane domains. The absolute expression profiles of these proteins that are temperature synchronized are also obtained from PlasmoDB.
Functional classification using Pfam v21.0
Since the P-loop NTPases are known to belong to functionally diverse groups therefore a domain analysis of dataset of 97 P. falciparum P-loop proteins is performed for functional classification of these proteins using Pfam v21.0 database of domains. The proteins are functionally correlated based on the domains present with E-values with a threshold of 10-2.
Secondary structure analysis of proteins
To perform secondary structure analysis of the P-loop domain of the dataset of 97 proteins, a representative dataset of 533 seed protein sequences is generated pertaining to Pfam accession ID: PF00004, PF07724, PF07726, PF04326 and PF07728. The secondary structure is predicted by the standalone version of PSIPRED v2.0, which is a simple and reliable method, incorporating two feed-forward neural networks that perform an analysis on output obtained from the initial run of PSI-BLAST at the cut-off E-value of 0.001 (Blast v2.2.4) . PSIPRED predicts the secondary structure for each residue and provides a confidence score for three types of secondary structures: helix, sheet and coil. The sequences are aligned according to the secondary structure for AAA+ proteins to classify them into classical AAA or AAA+ and other super-families based on the structural conformations and domain organizations. Multiple alignments are obtained using CLUSTALW (1.83) [14–16], which are manually refined to reflect the available structural information.
The phylogenetic relationships amongst various classes of proteins are determined using the Phylip 3.67 package . PROTDIST is used on the 97 sequences to calculate a distance matrix according to the Dayhoff PAM probability model . The computed distances represent the expected fraction of amino acid substitutions between each pair of sequences. The distance matrix is then used to estimate phylogenies using the neighbour joining (NJ) method. Bootstrapping is carried out using SEQBOOT (1,000 replicates for the PAM model of substitution). CONSENSE is used to compute the consensus tree by the majority rule method. The final unrooted tree diagram is generated using TreeView http://taxonomy.zoology.gla.ac.uk/rod/treeview.html.
Identification of orthologous protein sequences using OrthoMCL
To identify the possible drug targets in terms of orthology of proteins between P. falciparum and its human host and with other Apicomplexans, OrthoMCL algorithm is used , which performs the Markov Clustering (MCL) to group orthologs and paralogs across proteins of multiple organisms. The stand-alone version 1.3 of OrthoMCL is obtained from http://www.orthomcl.org/cgi-bin/OrthoMclWeb.cgi. Protein FASTA files of each of the genome is given as input to the algorithm. An all-against-all BLASTP analysis (at E value of 10-5) is carried out using OrthoMCL. The blast output, which describes genes paired by BLAST matches, the E-value, and the identity percentage and the related HSP information, is parsed to OrthoMCL. The evolutionary related proteins are interlinked in a similarity graph matrix. MCL http://micans.org/mcl/ is then invoked to split mega-clusters as an analogous process of manual review in COG construction (Clusters of Orthologous Groups). As a result, different clusters of orthologous proteins are created.
Results and discussion
Sequences with P-loop motifs: Generation of dataset
Using consensus sequences of Walker A (GxxGxGK [ST]), Walker B (hhhhDE) motifs and their variants along with other proteins annotated as P-loop NTPases (PlasmoDB), a dataset of 97 P. falciparum protein sequences is generated. These proteins showed variations in the length of the P-loop NTPase domains having insertions and repeat regions between Walker A and Walker B motifs. In addition, some of the proteins showed variations in the P-loop motifs compared to canonical forms, suggesting their divergence from other P-loop NTPases. These sequences are further analysed for protein domain organization, secondary structure and phylogenetic and orthologous relationships. A list of all the 97 proteins and their characteristics is provided in Additional file 2.
Functional classification based on domains and secondary structural analysis
In this class two myosin binding proteins of molecular motors, PFL1435c (myosin d) and PF13_0233 (myosin a) are identified. Unlike other myosins, PFL1435c contains long asparagine repeats between the Walker A and Walker B motifs. The orthologs of PF13_0233 protein are also found to be present in other Plasmodium species as well as in a closely related apicomplexan organism (Toxoplasma gondii); suggesting its evolutionary conservation. Four other kinases are also identified (MAL13P1.148, PF11_0416, PFE0175c and PFF0675c), that showed major variation in the P-loop domain organization; the P-loop domain of these proteins lack a classical Walker B motif. Although a total of 99 protein kinases have been identified in P. falciparum genome , only PF13_0334 is found to contain the classical P-loop NTPase fold.
The P. falciparum GTPases that have been classified within P-loop NTPases include PF11_0183, a nuclear binding protein and two transcription initiation and elongation factors, PFA0595c and PF13_0069. Based upon Pfam domain analysis along with their secondary structures, the two hypothetical proteins (PFF0810c and PF14_0052) are also classified as Kinase GTPases.
ASCE/RecA fold division
ABC transporters and SMC family
Helicases are enzymes that unwind duplex DNA or RNA coupled to nucleoside 5' triphosphate (NTP) binding and hydrolysis . Structural and sequence comparison of these proteins from different organisms have identified seven to nine short, conserved motifs called helicase signature motifs . Most of the known 3'-5' DNA helicases are members of SF1 or SF2. These two superfamilies have similar sets of conserved motifs that are responsible for coupling of ATP hydrolysis to DNA translocation and unwinding. However, sequence homology across SF1 and SF2 families is very weak and limited to the signature sequences, required for NTP binding (Walker A and B motifs) . ATP hydrolysis by SF1 helicases is stimulated only by ssDNA, whereas the ATPase activity of SF2 helicases is stimulated by both ssDNA and dsDNA . In the present study, seven P-loop NTPases in the P. falciparum genome are found to contain helicase, HA2 and DEAD/DEAH domain(s), characteristic of DNA/RNA helicases. The secondary structures of these helicases are dominated by helices towards the N-terminal, followed by regions having alternate α-helices and β-sheets. Multiple sequence alignments revealed that although the DEAH box helicases had high sequence conservation throughout the P-loop domain, PF08_0042 and PFC0440c had an 'I' → 'A' replacement in the DEAH box (Figure 4). In addition to some hydrophobic residue conservations among sequences, a sequence pattern 'I [LI]DE [AVIL] is found to be highly conserved. Downstream to the observed pattern, a Ser/Thr residue is found to be well-conserved amongst all the P. falciparum helicases.
Clamp loader/RFC clade
This clade is defined by the presence of two α-helices after strand 2 (of approximately equal size) that are packed against each other . The only representative of this family is PFL0150w. The N-terminus of the protein had rich low complexity regions and the ATPase domain had secondary structure similar to that of other AAA+ proteins (Figure 6).
ClpA/B ATPase clade
"Pre-sensor-1 β hairpin" (PS1BH) superclade
The remaining lineages of AAA+ namely HslU/ClpX/Lon, MCM, dynein/midasin and other relatives have been unified into a one large monophyletic group. The entire superclade is defined by the presence of an insert between the sensor-1 strand and the preceding helix known as "pre-sensor-1 β hairpin" . In this superclade, two distinct clades are identified, which are further divided into three protein families in the parasite genome.
The HslU/ClpX clade proteins contain one ATPase domain as compared to ClpA (with two ATPase domains). The ATPase domain of HslU is interrupted by an 'I' domain involved in the substrate binding [37, 38]. This clade is supported by an extended loop between strand-2 and helix-2. The HslU (ClpY) and ClpX ATPase interact with the protease partner HslV (ClpQ) and ClpA respectively to form multimeric protease degradation complex machineries as in case of ClpAP. Although ClpX ortholog is absent in P. falciparum, the ortholog of HslU (PFI0355c) annotated as ATP-dependent heat shock protein is identified. HslU orthologs are known in apicomplexans and kinetoplastids are predicted to be localized in the mitochondria or plastid . Sequence and structural analysis of PfHsIU confirmed the presence of three characteristic domains of HslU proteins, N-terminal domain (N-domain), Intermediate domain (I domain) and C-terminal domain (C-domain) . The HslU ortholog is an ATP binding regulatory subunit of prokaryotic proteasome complex with HslV/ClpQ threonine proteases. The ortholog of the prokaryotic HslV protease in the malaria parasite has been recently identified and is shown to be functionally important in the parasite . Both HslU and HslV lack any homolog in the vertebrate host and may be considered as a promising drug targets against the parasite. The Lon proteins from archea and bacteria define another family (LON family) within this clade. The PF14_0147 protein is identified as a P. falciparum Lon protease that contains the characteristic LAN domain and a Lon-protease domain flanking the AAA domain at the N- and C-termini, respectively.
Helix-2 insert clade
As the name suggests, the defining feature of the clade is an insert in helix-2 that folds into two β-strands . In the P. falciparum genome, the helix-2 insert clade is represented by Mini Chromosomal Maintenance proteins (MCMs) and dyneins, whereas other members of the clade such as NtrC, YifB and MoxR are not found in the parasite genome.
Dyneins are large molecular motor that transport various cellular cargo by "walking" along cytoskeletal microtubules towards the minus-end of the microtubule, which is usually oriented towards the cell center. Thus, they are called "minus-end directed motors". They contain six tandem AAA+ domains in the same polypeptide chain [9, 43]. The parasite genome contains seven representatives (average length of ~ 6000 amino acids) from this family out of which only four proteins (PF14_0626, MAL7P1.162, PF10_0224 and PF11_0240) are annotated at PlasmoDB. Three other proteins PFI0260c, PFL0115w and MAL7P1.89 annotated as hypothetical proteins at PlasmoDB, are identified as dyneins based upon the protein domain analysis. These proteins have two dynein heavy chain domains at their N-terminal named as DHC N-1 and DHC N-2 except in MAL7P1.89 and PF11_0240, which only have the DHC N-2 domain. The PFI0260c protein is also found to contain spectrin-like repeats.
Proteins belonging to the classical AAA clade contain highly conserved P-loop NTPase domain including SRH. The characteristic feature of the clade is the presence of an additional short helix immediately downstream of strand 2 in the P-loop domain . The AAA clade consists of all ATPases that originally are defined as the members of the AAA superfamily . The P. falciparum AAA proteins have been classified under following categories: metalloproteases, proteasomal subunits and 'D1 and D2' proteins. A conserved glycine residue at the N-terminal of the arginine finger is observed in all the members of this clade (Figure 7). Three P. falciparum proteins are identified as metalloproteases (pan-bacterial protein family) belonging to the M41 family of peptidases and proteases [45, 46]. PFL1925w has been annotated as cell-division protein FtsH whereas PF11_0203 and PF14_0616 have been annotated as hypothetical proteins (PlasmoDB). The domain analysis of these proteins showed that these hypothetical proteins contain a single AAA (Pfam domain id-PF00004) domain fused to the metalloprotease domain (Pfam domain id-PF01434) as in case of other FtsH proteins. Moreover, in all the three metalloproteases, a functional motif 'abXHEbbHbc' where 'a' is most often alanine or serine (instead of valine or threonine residues observed in metalloproteases of other organisms), 'b' is an uncharged residue (tyrosine or alanine here) and 'c' is a hydrophobic residue (leucine or isoleucine here) is observed. Thus both, domain and sequence-structure analysis, suggested that the two hypothetical proteins with characteristic features of the family may be functionally equivalent to FtsH metalloprotease. Upon performing the phylogenetic analysis based on their secondary structures, these three proteins tend to cluster together.
The 26S proteasomes complex is a component of the regulated protein degradation machinery in eukaryotic cell. The 19S regulatory component of the 26S proteasome complex consists of six distinct but closely related proteins (Rpt1–6) [47, 48]. These proteasomal ATPases contain tandem repeat of AAA module and are conserved throughout the archeao-eukaryotic branch [49, 50]. In the P. falciparum genome database, five proteins (PFD0665c, PF10_0081, PF11_0314, PF13_0033 and PF13_0063) have already been identified as Rpt homologues. Another protein PFL2345c, which is annotated as the tat-binding protein homolog, has also been found to be the part of this complex based on its sequence similarity and structural patterns. Secondary structure analysis shows that all six the proteins have classical helical and β-strand pattern (Figure 7). The 'D1 and D2' is another family that contains proteins with two AAA domains named as D1 and D2. This family includes N-ethylmaleimide-sensitive fusion protein (NSF), ATPase family g ene (AFG) and Cell Division Cycle 48 (CDC48) proteins [51, 52]. The NSF proteins play an important role in vesicle mediated protein trafficking in which the D1 AAA cassette is the active ATPase while D2 is nucleotide binding [47, 48]. The only NSF homologue identified in the malaria parasite is PFC0140c that contains the N-domain (essential for soluble NSF-attachment protein binding). The ATPase CDC48 family has two representatives in the P. falciparum genome, PF07_0047 and PFF0940c. An additional protein (MAL8P1.92) is observed, having similar sequence features and structural as that of known CDC48 proteins. It shows a significant sequence similarity of MAL8P1.92 with known CDC48 proteins along with the conserved secondary structural features such as the presence of an additional helix downstream of strand-2 in the ATPase domain. Phylogenetic analysis clustered proteins of proteasomal complex in a tight cluster close to another cluster of FtsH proteins (Figure 8).
MutS proteins/DNA mismatch repair proteins
AAA domain sequence and secondary structure based functional classification of hypothetical proteins
Absence of sequence similarity with any other known proteins is a major hurdle in functional classification of large number of P. falciparum hypothetical proteins. Martin  used hydropathy plots to identify novel membrane transporters from these hypothetical proteins of P. falciparum. The possible functional roles for few of P. falciparum hypothetical proteins are predicted based upon their grouping with other functionally annotated P-loop proteins in the phylogenetic analysis. The PF14_0126 protein is found to cluster with two functionally annotated RFC proteins and PFD0935c is observed to fall within the cluster of three MCM proteins and thus these proteins might have functional roles as RFC and MCM proteins respectively. Similarly PFF0810c and PF10_0099 are observed to cluster with SMC proteins, MAL13P1.13 with ABC transporters and PF14_0052 with ran/tc4 family proteins, suggesting their possible functional roles as SMC, ABC transporters and ran/tc4 proteins respectively. These hypothetical proteins have shown significant similarities at both the sequence and secondary structural levels. Thus, this approach may be extended to other organisms to classify and assign putative functions to many hypothetical proteins.
Identification of orthologous protein sequences using OrthoMCL
Firstly, 1,580 different orthologous groups are found between P. falciparum and Homo sapiens, consisting of 1,683 different proteins from the parasite. Interestingly, 37 P. falciparum P-loop NTPases have no ortholog in H. sapiens. These proteins include helicases, ABC transporters, Clp protease and heat shock protein. Out of these 37 proteins, 17 are annotated as hypothetical proteins. Together, this analysis offers opportunities to explore the potential of these proteins as novel drug targets without affecting the host.
The OrthoMCL analysis amongst six Plasmodium sp. namely P. falciparum, P. vivax, P. berghei, P. chabaudi, P. yoelii and P. knowlesi, identified 88 different P. falciparum P-loop NTPase proteins that have at least one ortholog in another malaria parasite species. The remaining nine NTPase proteins of P. falciparum are the proteins, which are unique to P. falciparum genome. These proteins include ABC transporter, 26S proteasome subunit Rpt3 and DNA replication licensing factor MCM2 in addition to hypothetical proteins. These proteins might be responsible for some dedicated pathways in the life-cycle of the parasite and thus can be of immense interest for further research that may provide clues to a number of unanswered questions in the parasite biology.
Since, there are few characteristic similarities among P. falciparum and plants; an attempt is made to identify orthologous of P. falciparum P-loop NTPase proteins in Arabidopsis thaliana a model organism to compare. A total of 57 parasite NTPase proteins have ortholog in A. thaliana genome. Four of these proteins are predicted to be targeted to the apicoplast of the P. falciparum. These are cell division cycle ATPase (PF07_0047), heat shock protein (PF11_0175), ATP-dependent transporter (PF14_0133) and a hypothetical protein (PF08_0063). Out of these four proteins, the ATP-dependent transporter protein has no human ortholog. The expression profile of this protein shows the peak expression at late-trophozoite and early schizont stages of the life-cycle. Together, this protein may be considered as a promising drug target.
The orthologous search is further extended to prokaryotes (Synechococcus sp., Mycobacterium tuberculosis, Escherichia coli and Staphylococcus aureus) where only 10 P. falciparum P-loop NTPases are observed to have orthologs in at least one of these prokaryotes. These 10 NTPases from the parasite showed sequence or structural similarity with bacterial ATPases such as multidrug resistance protein; heat shock protein and cell division protein FtsH protein (present in all eubacterial species). It strongly suggests that few unique processes of the parasite are governed by prokaryotic type mechanisms such as drug-resistance, cell-cycle or protein folding involving heat shock proteins. Some of these prokaryote like-parasite proteins may play crucial role in the parasite life cycle and can be studied as novel drug targets.
P-loop NTPases comprise one of the largest protein families with members present in all kingdoms of life. Numerous subgroups of the family are involved in diverse cellular functions. The functional roles of a number of families belonging to P-loop NTPases superfamily are still unknown in eukaryotes. The repertoire of the P-loop NTPase has not been identified and classified in P. falciparum. The challenge of studying and classifying these NTPases further increases in P. falciparum due to low sequence similarity and unique features of the genome such as extended insertions and repeats. In the present study, a systematic classification of the P-loop NTPases in P. falciparum genome is carried out, that provided information on their function, classification, phylogenetic and orthologous relationships amongst various protein families and organisms. Variations in critical residues within the conserved regions as well as long insertions are observed in the P-loop NTPase domain for most of the P. falciparum NTPases suggesting that the parasite has evolved constantly to sustain inspite of the mutations/variations in these imperative regions. The study provided an understanding of the P-loop NTPases, especially in terms of their structural and functional relationships. The proteins with similar functional roles are observed to have similar sequence and structure pattern of P-loop domain. Based on this, putative functional roles for 14 hypothetical proteins are predicted. This is one of the key findings of the study pertaining to the fact that most of P. falciparum proteins are not homologous to any other eukaryotic protein and have been annotated as hypothetical proteins. Therefore, elucidation of putative roles of these proteins that are unique to the parasite may provide leads to identify novel drug targets. The sequence orthology based studies are found to be useful in identifying P-loop NTPases either similar to prokaryotic origin or restricted to Plasmodium species. Such P-loop NTPases involved in important physiological pathways may lead to identification of new drug targets. It must be emphasized that the current study demonstrates the possible achievements of a computational analysis and is a preliminary investigation. Experimental evidence to explore the role of these genes is thus required. It becomes mandatory in the case of P. falciparum where new functional roles have been predicted for a significant number of hypothetical proteins inspite of very low levels of sequence similarity. Overall the study provides us new leads in investigating the functions and biology of P. falciparum P-loop NTPases.
DG and MKK are supported by research fellowship from Council of Scientific and Industrial Research, India. The research in lab of AM is supported by Department of Biotechnology, Govt. of India.
- Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005, 434: 214-217.PubMed CentralView ArticlePubMed
- McConkey AG, Pinney WJ, Westhead RD, Plueckhahn K, Fitzpatrick BT, Macheroux P, Kappes B: Annotating the Plasmodium genome and the enigma of the shikimate pathway. Trends Parasitol. 2004, 20: 60-65.View ArticlePubMed
- Koonin EV, Aravind L: The NACHT family – a new group of predicted NTPases implicated in apoptosis and MHC transcription activation. Trends Biochem Sci. 2000, 25: 223-224.View ArticlePubMed
- Saraste M, Sibbald PR, Wittinghofer A: The P-loop – a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990, 15: 430-434.View ArticlePubMed
- Vetter IR, Wittinghofer A: Nucleoside triphosphate-binding proteins: different scaffolds to achieve phosphoryl transfer. Q Rev Biophys. 1999, 32: 1-56.View ArticlePubMed
- Mogk A, Dougan D, Weibezahn J, Schlieker C, Turgay K, Bukau B: Broad yet high substrate specificity: the challenge of AAA+ proteins. J Struct Biol. 2003, 146: 90-98.View Article
- Walker JE, Saraste M, Runswick MJ, Gay NJ: Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP requiring enzymes and a common nucleotide binding fold. EMBO J. 1982, 1: 945-951.PubMed CentralPubMed
- Milner-White EJ, Coggins JR, Anton IA: Evidence for an ancestral core structure in nucleotide-binding proteins with the type A motif. J Mol Biol. 1991, 221: 751-754.View ArticlePubMed
- Neuwald AF, Aravind L, Spouge JL, Koonin EV: AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 1999, 9: 27-43.PubMed
- Fröhlich KU: An AAA family tree. J Cell Sci. 2001, 114: 1601-1602.PubMed
- Frickey T, Lupas AN: Phylogenetic analysis of AAA proteins. J Struct Biol. 2004, 146: 2-10.View ArticlePubMed
- Lupas AN, Martin J: AAA proteins. Curr Opin Struct Biol. 2002, 12: 746-753.View ArticlePubMed
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402.PubMed CentralView ArticlePubMed
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997, 25: 4876-4882.PubMed CentralView ArticlePubMed
- Notredame C, Higgins DG, Heringa J: T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217.View ArticlePubMed
- Wolf YI, Rogozin IB, Kondrashov AS, Koonin EV: Genome alignment, evolution of prokaryotic genome organization, and prediction of gene function using genomic context. Genome Res. 2001, 11: 356-372.View ArticlePubMed
- Felsenstein J: PHYLIP Phylogeny Inference Package 3.5. 1993, Department of Genetics, The University of Washington, Seattle, WA
- Dayhoff M: Atlas of Protein Sequence and Structure. 1978, National Biomedical Research Foundation, Washington, DC
- Li L, Stoeckert CJ, Roos DS: OrthoMCL: Identification of ortholog groups for eukaryotic genomes. Genome Res. 2003, 13: 2178-89.PubMed CentralView ArticlePubMed
- Leipe DD, Wolf YI, Koonin EV, Aravind L: Classification and evolution of P-loop GTPases and related ATPases. J Mol Biol. 2002, 317: 41-72.View ArticlePubMed
- Leipe DD, Koonin EV, Aravind L: Evolution and classification of P-loop kinases and related proteins. J Mol Biol. 2003, 333: 781-815.View ArticlePubMed
- Anamika , Srinivasan N, Krupa A: A Genomic Perspective of Protein Kinases in Plasmodium falciparum. Proteins: Structure, Function, and Bioinformatics. 2005, 58: 180-189.View Article
- Dean M, Rzhetsky A, Allikmets R: The Human ATP-Binding Cassette (ABC) Transporter Superfamily. Genome Research. 2001, 11: 1156-1166.View ArticlePubMed
- Klokouzas A, Barrand MA, Hladky SB: Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells. Eur J Biochem. 2001, 268: 6569-6577.View ArticlePubMed
- Bozdech Z, VanWye J, Haldar K, Schurr E: The human malaria parasite Plasmodium falciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell. Mol Biochem Parasitol. 1998, 97: 81-95.View ArticlePubMed
- Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The Transcriptome of the Intraerythrocytic Developmental Cycle of Plasmodium falciparum . PLoS Biol. 2003, E5-1
- Ginsburg H: Progress in in-silico functional genomics: the malaria Metabolic Pathways database. Trends Parasitol. 2006, 22: 238-40.View ArticlePubMed
- Bodó A, Bakos E, Szeri F, Váradi A, Sarkadi B: The role of multidrug transporters in drug availability, metabolism and toxicity. Toxicol Lett. 2003, 140–141: 133-143.View ArticlePubMed
- Lohman TM, Bjomson KP: Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem. 1996, 65: 169-214.View ArticlePubMed
- Gorbalenya AE, Koonin EV: An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication. J Mol Evol. 2003, 28: 256-268.View Article
- Koonin EV, Dolja VV: Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol. 1993, 28: 375-430.View ArticlePubMed
- Singleton MR, Wigley DB: Modularity and specialization in superfamily 1 and 2 helicases. J Bacteriol. 2002, 184: 1819-1826.PubMed CentralView ArticlePubMed
- Vale RD: AAA proteins. Lords of the ring. J Cell Biol. 2000, 150: F13-9.PubMed CentralView ArticlePubMed
- Hanson IP, Whiteheart SW: AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol. 2005, 6: 519-29.View ArticlePubMed
- Iyer LM, Leipe DD, Koonin EV, Aravind L: Evolutionary history and higher order classification of AAA+ ATPases. J Struct Biol. 2004, 146: 11-31.View ArticlePubMed
- Hoskins JR, Sharma S, Sathyanarayana BK, Wickner S: Clp ATPases and their role in protein unfolding and degradation. Adv Protein Chem. 2001, 59: 413-429.View ArticlePubMed
- Kwon AR, Kessler BM, Overkleeft HS, McKay DB: Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU, a prokaryotic homolog of the eukaryotic proteasome. J Mol Biol. 2003, 330: 185-195.View ArticlePubMed
- Song HK, Bochtler M, Azim MK, Hartmann C, Huber R, Ramachandran R: Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV. Biophys Chem. 2003, 100: 437-52.View ArticlePubMed
- Couvreur B, Wattiez R, Bollen A, Falmagne P, Le Ray D, Dujardin JC: Eubacterial HslV and HslU subunits homologs in primordial eukaryotes. Mol Biol Evol. 2002, 19: 2110-2117.View ArticlePubMed
- Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD, Huber R: The structures of HsIU and the ATPdependent protease HsIU-HsIV. Nature. 2000, 403: 800-805.View ArticlePubMed
- Ramasamy G, Gupta D, Mohmmed A, Chauhan VS: Characterization and localization of Plasmodium falciparum homolog of prokaryotic ClpQ/HslV protease. Mol Biochem Parasitol. 2007, 152: 139-48.View ArticlePubMed
- Koonin EV: A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res. 1993, 21: 2541-2547.PubMed CentralView ArticlePubMed
- Mocz G, Gibbons IR: Model for the motor component of dynein heavy chain based on homology to the AAA family of oligomeric ATPases. Structure. 2001, 9: 93-103.View ArticlePubMed
- Confalonieri F, Duguet M: A 200-amino acid ATPase module in search of a basic function. Bioessays. 1995, 17: 639-650.View ArticlePubMed
- Rawlings ND, Barrett AJ: Evolutionary families of metallopeptidases. Methods Enzymol. 1995, 248: 183-228.View ArticlePubMed
- Rawlings ND, Barrett AJ: MEROPS: The peptidase database. Nucleic Acids Res. 1999, 27: 325-31.PubMed CentralView ArticlePubMed
- Ogura T, Wilkinson AJ: AAA+ superfamily ATPases: common structure-diverse function. Genes Cells. 2001, 6: 575-97.View ArticlePubMed
- Ogura T, Whiteheart SW, Wilkinson AJ: Conserved arginine residues implicated in ATP hydrolysis, nucleotidesensing, and inter-subunit interactions in AAA and AAA+ ATPases. J Struct Biol. 2004, 146: 106-112.View ArticlePubMed
- Beyer A: Sequence analysis of the AAA protein family. Protein Sci. 1997, 6: 2043-2058.PubMed CentralView ArticlePubMed
- Swaffield JC, Purugganan MD: The evolution of the conserved ATPase domain (CAD): reconstructing the history of an ancient protein module. J Mol Evol. 1997, 45: 549-63.View ArticlePubMed
- Lee YJ, Wickner RB: AFG1, a new member of the SEC18- NSF, PAS1, CDC48-VCP, TBP family of ATPases. Yeast. 1992, 8: 787-90.View ArticlePubMed
- Ye Y, Meyer HH, Rapoport TA: The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature. 2001, 414: 652-656.View ArticlePubMed
- Wood MA, McMahon SB, Cole MD: An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc. Mol Cell. 2000, 5: 321-330.View ArticlePubMed
- Kurokawa Y, Kanemaki M, Makino Y, Tamura TA: A notable example of an evolutionary conserved gene: studies on a putative DNA helicase TIP49. DNA Seq. 1999, 10: 37-42.View ArticlePubMed
- Martin RE, Henry RI, Abbey JL, Clements JD, Kirk K: The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum . Genome Biol. 2005, 6: R26-PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.