Systematic In Silico Discovery of a Conserved Tubulin-Folding Chaperone Network in Plasmodium falciparum: Implications for Microtubule Homeostasis and Antimalarial Targeting

1. Introduction

Microtubules are highly dynamic polymers that are formed as a result of polymerization and depolymerization of tubulin dimers, and are essential molecules for maintaining cytoskeletal architecture, intracellular transport, and cell division in eukaryotes [1,2]. The biogenesis of αβ-tubulin heterodimers is a highly regulated process as neither tubulin can fold autonomously, instead they require a committed multistep pathway involving a conserved network of molecular chaperones to achieve their functional conformation [3,4,5]. The pathway is initiated as soon as tubulin emerges from the ribosome in an unfolded state, and is captured by the chaperone prefoldin which subsequently transfer it to cytosolic chaperonin TRiC for proper folding [3,5,6]. TRiC encapsulates tubulin within its chamber as it undergoes a closed conformation upon ATP hydrolysis [6,7]. Post this, polymerization-competent αβ-tubulin heterodimers are formed in a highly coordinated series of reactions mediated by a specific group of tubulin-specific chaperones (TBCs), also known as tubulin-folding (TBCA–TBCE), and the small GTPase Arl2 [4,5,8]. Altogether, coordinated activity of these chaperones along with Arl2 provide quality-control machinery for the precise regulation of αβ-tubulin heterodimer homeostasis [4,5,8].

Early biochemical reports suggested that TBCs act sequentially to stabilize αβ-tubulin intermediates and facilitate hetero-dimerization via GTP-dependent hydrolysis [9,10,11]. Quasi-native folding intermediates of αβ-tubulin are captured and stabilized by free exchange between TBCA and TBCD (in case of β-tubulin), and between TBCB and TBCE (in case of α tubulin), resulting in the formation of TBCD/β and TBCE/α, respectively, which latter form a supercomplex (TBCD/α/ TBCD/β) [9,10]. Post this, TBCC interacts with supercomplex and triggers GTP hydrolysis in the E-site thereby releasing newly formed α/β tubulin heterodimers. Following spontaneous E-site exchange with GTP, α β heterodimers acquire competency to form the microtubule polymer [10,11]. The tubulin-specific chaperones thus act as a tubulin assembly machine regulating not only de novo folding but also the recycling and turnover of the tubulin heterodimer [4,5,11].

A recent report by Seong et. al, proposed a new molecular mechanism by which TBCs maintain tubulin homeostasis [12]. The authors used cryo-electron microscopy to determine structures of the human TBCs at six stages under defined nucleotide conditions. The data suggest that TBCD, TBCE, and Arl2 form a cage-like TBC-DEG complex that encapsulates α- and β-tubulin in partially assembled states [12]. TBCC subsequently act as a molecular clamp and trigger the conformational rearrangement that allows α- and β-tubulin interfaces for productive GTP hydrolysis that ultimately leads to formation of α β heterodimers [12]. The authors also highlighted TBCs as central regulators capable of both promoting assembly and driving disassembly of α β heterodimers under specific cellular conditions, instead of merely functioning as molecular chaperones [12]. This dual role underscore significance of TBCs in maintaining tubulin homeostasis, particularly in organisms where microtubule dynamics are highly responsive to stress and developmental cues [12].

In light of the above facts and several other studies in mammalian models and plants, TBCs are considered evolutionarily conserved essential molecules required for the biogenesis, maintenance, and degradation of αβ tubulin heterodimers [5,13,14,15]. While the canonical tubulin-specific chaperones (TBCs A–E) are well-known in most eukaryotes, the information of these specialized proteins within the Plasmodium falciparum genome remains totally unidentified. Tubulins form essential building blocks for several specialized microtubule-based structures that govern malaria parasite complex life cycle in human and mosquito host [16,17,18]. These structures are vital for maintaining parasite-specific shapes, driving unique forms of gliding motility, and ensuring successful host invasion as well as transmission [16,17,18]. Microtubule function is governed not only by the assembly of α- and β-tubulin heterodimers but also by a diverse repertoire of post-translational modifications (PTMs), collectively referred to as the "tubulin code" [19,20]. Consequently, any change in tubulin PTMs can influence the structural integrity, stability, and interaction network of mature tubulin heterodimers, thereby affecting downstream cytoskeletal organization [19,20]. This suggest that the efficiency of tubulin folding and maturation mediated by TBCs may determine the availability of correctly assembled tubulin substrates for subsequent post-translational modification. Since TBCs play a pivotal role in the tubulin biogenesis pathway and considering the significance of tubulins in parasite biology, the present study represents the first comprehensive identification and in silico characterisation of the tubulin-specific chaperone (PfTBC) family in the P. falciparum genome. Using an integrative bioinformatics approach, we identified putative PfTBC proteins and systematically analysed their sequence features, conserved domains, structural organisation, evolutionary relationships, expression profiles, and predicted interaction networks. By establishing a foundational framework for understanding the tubulin-folding machinery in P. falciparum, this study provides novel insights into parasite cytoskeletal biology and highlights PfTBCs as previously unexplored components of the parasite proteostasis network. Furthermore, our study suggest PfTBCs as promising drug targets for future functional studies and the development of selective antimalarial strategies targeting parasite-specific mechanisms of microtubule homeostasis.

2. Materials and Methods

2.1. Sequence Retrieval and Domain Organization Analysis

The FASTA sequences of PfTBCs were retrieved from the PlasmoDB database [21]. Putative candidate genes were subjected to conserved domain analysis using the Conserved Domain Database (CDD) [22] and InterProScan [23] to verify the presence of hallmark tubulin-specific chaperone (TBC) domains. BLASTp against specific organisms was used to identify homologues of PfTBCs [24], whereas multiple sequence alignment and phylogenetic analysis were performed using Clustal Omega [25].

2.2. Gene Expression Analysis

Transcriptomic and proteomic data for identified PfTBCs were retrieved from publicly available datasets deposited in PlasmoDB [21]. Heat maps and expression plots were generated using GraphPad Prism to visualize relative expression trends, with expression values log₂-transformed where necessary for comparative analysis.

2.3. Essentiality Profiling and Protein-Protein Interaction Network Analysis

Essentiality of putative tubulin-specific chaperone (TBC) genes in Plasmodium falciparum was assessed using phenotype-based functional genomics data retrieved from PlasmoDB [21] and the PhenoPlasm database [26], which integrate results from large-scale genetic screens, including saturation transposon mutagenesis and targeted gene disruption studies. Protein–protein interaction networks for identified PfTBCs were constructed using the STRING database [27], incorporating both experimentally validated and predicted interactions, with a confidence score threshold of ≥0.4. Gene Ontology enrichment analyses were performed to identify key interacting partners involved in tubulin folding, cytoskeletal organization, and cell division.

2.4. Structure Modelling and Complex Studies

Protein–protein docking calculations were performed using the PIPER (Protein–Protein Interaction Prediction Engine) algorithm integrated within Schrödinger Maestro (Schrödinger Release 2023-4) [28,29]. Three-dimensional structures of all proteins were derived from Plasmodium falciparum protein sequences obtained from PlasmoDB [21] and predicted using AlphaFold2 [30]. Rigid-body docking was performed without constraints or restraints, utilizing default PIPER parameters. Post-docking processing included clustering analysis based on RMSD criteria and energy ranking. Binding interface analysis was conducted using Maestro's integrated tools: hydrogen bond detection (distance cutoff ≤3.2 Å), salt bridge analysis (distance ≤4.0 Å), hydrophobic contact identification (distance ≤4.0 Å), and buried surface area (SASA) calculations. Surface complementarity was calculated using shape-based complementarity metrics. Van der Waals clash analysis identified close contacts. All calculations employed standard pH 7.4 conditions. Structural superimposition of the protein models was performed using the align command in PyMOL [31].

3. Results

3.1. Identification and Comparative Evolutionary Analysis of TBCs in the Malaria Parasite

In malaria parasite Plasmodium falciparum, the folding and regulation of tubulin is of particular interest, as microtubules drive essential processes including schizogony, merozoite morphogenesis, and gametocyte development. Parasite-specific adaptations in tubulin cofactors may represent unexplored vulnerabilities for antimalarial intervention, especially because the parasite’s cytoskeletal machinery diverges significantly from that of humans. However, identification and comprehensive characterization of TBCs in parasites remains totally unexplored, and understanding their structural and functional divergence may contribute to fill the key knowledge gaps and uncover novel therapeutic targets. Uniprot [32] was used to search P. falciparum genome for presence of tubulin specific chaperones genes in P. falciparum (PfTBCs) and three genes viz. PF3D7_0109400 (Uniprot ID: Q8I250_PLAF7), PF3D7_0906910 (Uniprot Id: Q8I381_PLAF7) and PF3D7_1015700 (Uniprot Id: C6S3C6_PLAF7) for Tubulin-specific chaperone A, Tubulin-specific chaperone, and tubulin binding cofactor c respectively were identified. Plasmodb database [21] was then searched using their Plasmodb IDs (PF3D7_0109400; PF3D7_0906910 and PF3D7_1015700), and putative proteins were identified. Domain analysis using CDD database [22] demonstrated the presence of a domain characteristic of TBCA, TBCB, TBCC family in PF3D7_0109400, PF3D7_0906910 and PF3D7_1015700 respectively (Figure 1 A). Interestingly, our search in Uniprot provided a hit named PI3K/PI4K catalytic domain-containing protein (Uniprot Id: Q8IKD7_PLAF7; Plasmodb Id: PF3D7_1470100) and sequence analysis using InterPro database [23] demonstrated centrally located beta tubulin cofactor D domain in this protein (Figure 1 A). To look for the presence of TBCE in Pf, organism-specific Blastp search [24] was performed using human TBCE sequence that provided most reliable homologue in Pf. Detailed domain organization analysis using CDD clearly demonstrated the presence of C terminal domain characteristic of TBCE protein family (Figure 1 A). The presence of PfTBCs homologues in other species was next explored. Organism-specific Blastp search using PfTBCs sequences provided their most reliable homologues in Homo sapiens, Sacharomyces cerevesisia, Drosophila melanogaster, Mus Musculus, Arabidopsis thaliana etc. All TBC sequences were subjected to multiple sequence alignment, and the resulting alignment was used to construct a phylogenetic tree to infer evolutionary and sequence-level relationships among the TBCs (Figure1 B).

3.2. Expression Analysis of TBCs in Asexual and Sexual Stages

To look into the mechanistic insights of PfTBCs family in malaria parasite, we next explored their expression analysis by digging the transciptomics datasets from Plasmodb [21] during the asexual and sexual stages. Interestingly, we observed upregulation in all PfTBCs expression during the intraerythrocytic development cycle of malaria parasite, with notable expression peaks at the trophozoite stage, suggesting its possible role in development-related processes (Figure 2 A). Moreover, PfTBCs show prominent increase in expression, particularly during gametocytogenesis, with male gametocytes showing significantly higher transcripts compared to females (Figure 2 B). This stage-specific variation indicates their potential role in sexual differentiation processes. Overall, these data underscore the gene's essential role in parasite development and adaptation.

3.3. Essentiality Profiling of PfTBCs and Their Interaction Networks in the Malaria Parasite

We next explored essentiality profiling of PfTBCs using PhenoPlasm database [26]. Interestingly, all PfTBCs were found to be essential for survival of the parasite, underscoring their importance in parasite biology, and thereby making them promising leads for development as antimalarial agents. Further we explored the interaction network of PfTBCs using STRING protein–protein interaction database [27] and found tubulin alpha chain and beta chain as major hits for PfTBCs (Figure 3 i-v). These observations provide compelling evidence that they are involved at the molecular level in assisting assembly and disassembly of tubulin heterodimers. Interestingly, we identified Plasmodium falciparum prefoldin subunits (PfPFDs) as interacting partners of PfTBCs, consistent with the established model that PfPFDs bind newly synthesized PfTBCs and facilitate their transfer to the chaperonin TRiC complex for proper folding (Figure 3 i-v).

3.4. In Silico Interaction Interface Analysis of PfTBC-B with Pf α-Tubulin and PfTBC-D with Pf β-Tubulin

Rigid-body protein-protein docking of PfTBC-B with Pf α-Tubulin was performed using PIPER [30] that generated 30 distinct binding poses. The top-ranked pose exhibited PIPER energy −748.314 kcal/mol (score −194.711) from the largest cluster (n = 63) (Table 1). Energy values ranged from −1035.063 to −748.314 kcal/mol. Cluster size analysis revealed high-ranking poses derived from clusters containing 19–63 members, indicating reproducibility of the predicted binding orientation. Binding interface analysis identified 78 residue-residue interactions (≤4.0 Å). One prominent salt bridge anchors the PfTBC-B - Pf α-Tubulin complex: Asp102–Lys401 (2.2 Å distance, 100% occupancy, and complementarity 0.51) (Table 2; Figure 4). One pi-stacking interaction provides enhanced aromatic recognition: Phe14–Phe404 (2.7 Å, 100% occupancy, complementarity 0.87, exceptional burial at Phe404: 99.6%) (Table 2; Figure 4). Extensive hydrophobic networks are organized into three regions: N-terminal aromatic cluster (Phe14, Lys17, Trp19 of PfTBC-B engaging Phe404, Trp407, His406 of Pf α-Tubulin; mean burial 59.7% PfTBC-B, 87.2% Pf α-tubulin), middle hydrophobic region (His40, Thr39, Tyr38, Tyr99 of PfTBC-B contacting Trp407, His406, Val181, Arg402 of Pf α-tubulin ; mean complementarity 0.74, burial 82.7%), and C-terminal region (Ser245, Asn244, Lys187 of PfTBC-B engaging Arg339 of Pf α-tubulin). Surface complementarity analysis revealed mean score 0.69 ± 0.18 with 66.7% of interactions >0.80. Mean buried SASA: PfTBC-B 47.8%, Pf α-tubulin 61.2%.

For PfTBC-D - Pf β-Tubulin docking PIPER generated 30 binding poses with exceptional reproducibility. The top-ranked pose exhibited PIPER energy −753.113 kcal/mol (score 11.624) from the largest conformational cluster observed across all studies (n = 72) (Table 3). Energy values ranged from −1001.708 to −709.439 kcal/mol. Cluster sizes ranged from 72 to 5 members. The exceptional cluster size of 72 provides unprecedented statistical validation of the predicted binding mode. Interface analysis identified 62 residue-residue interactions (≤3.9 Å). Two salt bridges anchor the PfTBC-D - Pf β-Tubulin complex with directional specificity: Glu439–Arg1613 (1.9 Å, 100% occupancy, complementarity 0.79, burial 84.8% / 86.4%) and Glu438–Lys1534 (2.5 Å, 100% occupancy, complementarity 0.86, burial 53.2% / 39.0%) (Table 4; Figure 4). Additional hydrogen bonds provide supplementary stabilization: Glu435–Lys1611 (2.2 Å, salt bridge + H-bond, complementarity 0.80, burial 87.4% / 62.3%) and Glu432–Lys1393 (2.5 Å, H-bond, complementarity 0.56, burial 86.9% / 24.2%) (Table 4; Figure 4). One van der Waals clash (Tyr425–Gln1618, 1.7 Å, complementarity 0.82, burial 93.3% / 39.5%) exhibits optimal geometric fit. Three functional regions stabilize the interface: acidic anchor cluster (Glu432–Glu444, mean burial 51.7% PfTBC-D, 77.6% Pf β-tubulin, 18 interactions), aromatic-hydrophobic secondary region (Phe436, Gln424, Tyr425, Phe260, mean complementarity 0.83, burial 80.4% / 72.5%, 20 interactions), and tertiary contact cluster (Pro346, Ile345, Trp344, 8 interactions) (Table 4; Figure 4). Surface complementarity analysis revealed mean score 0.71 ± 0.27 with 69.4% of interactions >0.80—exceptional geometric specificity. Mean buried SASA: PfTBC-D 51.4%, Pf β-tubulin 54.8%.

3.5. Comparison of PfTBCs with Human Counterparts

Comparative analysis of the five Plasmodium falciparum TBCs (PfTBCA–PfTBCE) with their corresponding human orthologs was conducted with a perspective of studying their candidature for anti-malarial drug targets. Pairwise sequence alignment revealed that PfTBCs exhibit considerable divergence at the primary sequence level, with only 25%, 24.7%, 17.3%, 10.8% and 7.8% sequence identity of PfTBC-A, PfTBC-B, PfTBC-C, PfTBC-D, PfTBC-E respectively with their corresponding human counterparts (Table 5). These data show that PfTBCs represent attractive candidates for antimalarial drug discovery owing to their essential role in microtubule biogenesis and their sequence divergence from the corresponding human proteins which provide opportunities for the rational design of selective inhibitors that can target parasite tubulin-folding machinery while minimizing cross-reactivity with the human homologs.

We next performed structural superposition of the PfTBCs with their respective human homologues that revealed conservation of the overall three-dimensional architecture despite pronounced sequence divergence (Figure 5). The superimposed structures depicted significant overlap of the central structural cores, suggesting preservation of the characteristic fold across the orthologous proteins. In contrast, conformational differences were predominantly localized to surface-exposed loops, terminal extensions, and flexible peripheral regions. Although structural superposition demonstrated conservation of the overall fold, PfTBC-B, PfTBC-E exhibited relatively high RMSD values (~15 Å, ~ 10 Å respectively), indicating pronounced conformational divergence in domain orientations, loop regions and terminal extensions and such parasite-specific structural differences may contribute in designing selective inhibitors that can preferentially target the Plasmodium proteins while minimizing interactions with their human counterparts. Overall, these findings suggest that the structural framework required for tubulin-folding activity is conserved, whereas parasite-specific structural variations may provide opportunities for selective therapeutic targeting.

3.6. Role of PfTBCs in Artemisinin Resistance

Several observations from recent omics studies identify expression of TBCs including PfTBCA in transcriptomic and proteomic datasets associated with parasite quiescence and artemisinin-related studies [33,34,35,36]. TBCA has also been reported in the translatome of quiescent stage V gametocytes [34], indicating active translation during a metabolically restrained developmental stage. This gene has also appeared in transcriptomic datasets associated with investigations of artemisinin resistance [35,36], although it has not yet undergone functional characterization. These observations identify PfTBC-A as a candidate deserving further investigation in the biology of artemisinin resistant parasites.

4. Discussion

Microtubule biogenesis involve several interconnected steps that regulate diverse processes including mitotic spindle formation, chromosome segregation, host cell invasion, merozoite assembly, and gametogenesis, thereby facilitate survival and transmission of Plasmodium falciparum [16,37,38,39]. The precise folding of α- and β-tubulin into assembly-competent heterodimers is mediated by a conserved tubulin-folding machinery involving the chaperonin TRiC/CCT complex and tubulin-binding chaperones [3,4,5,6,7,8,9,10,11,12]. Although this pathway has been extensively characterized in higher eukaryotes, its composition and functional organization in malaria parasites have remained largely unexplored [4,5,10,11,12]. Here, we attempted to identify all five TBCs (TBCA-E) in Plasmodium falciparum and provide the first comprehensive in silico characterization, expression profiling, and their interaction analysis with their partner proteins, establishing a foundation for understanding their contribution to parasite biology.

Our bioinformatic analysis identified putative orthologues of all five canonical tubulin-binding cofactors in P. falciparum. Conserved domain analysis confirmed the presence of characteristic TBCA, TBCB, TBCC, TBCD, and TBCE domains, suggesting that the parasite harbors the molecular machinery required for tubulin biogenesis. Comparative evolutionary analysis revealed that PfTBCs cluster with their respective orthologues from diverse eukaryotes while remaining evolutionarily distinct. This conservation is consistent with the essential role of tubulin-folding cofactors across eukaryotic organisms [5,13,14,15]. At the same time, the divergence observed suggest specific adaptations that may have evolved to assist the unusual cell division mechanisms of apicomplexan parasites [17,18]. Unlike mammalian cells, P. falciparum undergoes schizogony, a specialized form of endo-mitosis involving multiple rounds of asynchronous nuclear division prior to cytokinesis [16,37]. Moreover, sexual differentiation involves extensive cytoskeletal remodeling, particularly during male gametogenesis, where rapid microtubule polymerization facilitates the formation of eight flagellated microgametes within minutes [38,39,40]. These unique processes are likely to impose distinct regulatory requirements on the parasite tubulin-folding machinery that may be responsible for evolutionary divergence of PfTBCs from their mammalian counterparts.

Transcriptomic analysis revealed expression of PfTBCs during the asexual blood stages, with peak transcript abundance during the trophozoite stage. This developmental stage is depicted by intense metabolic activity, rapid protein synthesis, DNA replication, and preparation for schizogony [41,42], all of which require extensive cytoskeletal remodeling [16,37,38]. Up-regulated expression of PfTBCs during this stage underscores the importance of increased demand for correctly folded tubulin heterodimers necessary for mitotic spindle assembly and daughter merozoite formation. Interestingly we found pronounced up-regulation of PfTBC transcripts during gametocytogenesis, especially in male gametocytes. Male gamete formation represents one of the fastest microtubule assembly events known in eukaryotes, requiring axoneme formation and rapid nuclear division immediately followed by activation within the mosquito midgut [38,39,40]. The up-regulated expression of PfTBCs during this stage strongly suggests that these proteins contribute to the efficient generation of assembly-competent tubulin required for axoneme biogenesis. Functional significance of the PfTBC family was further supported by essentiality profiling using PhenoPlasm, which classified all identified PfTBC genes as essential for parasite survival. The essential nature of all PfTBCs suggests that any inhibition to the function of these proteins can severely disrupt tubulin-folding pathway ultimately effecting parasite viability, thereby highlighting these proteins as promising antimalarial drug targets. Importantly, unlike tubulin, which is highly conserved between parasites and humans [43] and therefore poses challenges for selective drug targeting, TBCs exhibit greater sequence divergence while maintaining conserved biological function. Such divergence may provide opportunities for the development of parasite-selective inhibitors with reduced host toxicity. Consequently, PfTBCs may represent attractive multi-stage targeting candidates owing to their role during intra-erythrocytic stages as well as gametocyte developmental stages. This suggest that PfTBCs can be exploited not only for inhibiting asexual parasite proliferation but also for disrupting malaria transmission by impairing gamete development. Moreover, the recurrent detection of PfTBC-A across independent transcriptomic, proteomic, and translatomic studies [17,37,38,39] suggests that this tubulin-specific chaperone may have a broader role in parasite adaptation. Although no direct functional evidence currently links PfTBC-A to artemisinin resistance, its consistent expression in quiescent parasites and stage V gametocytes [17,37,38,39] indicates that it remains an active component of the cellular machinery during metabolically restrained states. This indicates that PfTBC-A may contribute to parasite survival, recovery from dormancy, and the emergence or maintenance of artemisinin resistance, suggesting this protein as a potential therapeutic target for overcoming parasite persistence and improving antimalarial efficacy. If tubulin-specific chaperones prove to contribute to parasite survival following artemisinin exposure, the implications would extend beyond understanding resistance mechanisms. Components of the tubulin-folding machinery could represent attractive therapeutic targets capable of sensitizing resistant parasites to existing antimalarial drugs. Pharmacological disruption of tubulin-specific proteostasis, particularly when combined with artemisinin derivatives, might impair recovery from dormancy and reduce parasite survival without directly targeting highly conserved tubulin polymers themselves. Such strategies may offer opportunities to overcome resistance while minimizing selective pressure on existing drug targets.

Collectively, these findings significantly expand our understanding of microtubule biogenesis in P. falciparum. While microtubules have long been recognized as attractive antimalarial targets, most therapeutic efforts have focused on directly disrupting tubulin polymerization [43,44,45]. Targeting the upstream tubulin-folding machinery represents an alternative strategy that may compromise microtubule formation before functional polymers are assembled. Figure 6 shows proposed model depicting the mechanism of α/β-tubulin biogenesis pathway. The model highlights the role of PfTBCs and their potential as parasite-specific drug targets in Plasmodium falciparum. Given the essentiality of PfTBCs, their developmental regulation, and their evolutionary divergence from human homologues, these proteins constitute promising candidates for therapeutic intervention. Future studies should focus on biochemical characterization of individual PfTBCs, determination of their three-dimensional structures, identification of interacting partners through experimental proteomics, and functional validation using conditional genetic approaches. Such investigations will provide mechanistic insight into parasite-specific tubulin biogenesis and may facilitate the development of novel antimalarial agents targeting this previously unexplored pathway.