In silico analysis of a Skp1 protein homolog from the human pathogen E. histolytica

Abstract

SCF complex consisting of Skp1, Cullins, F-box proteins, is the largest family of E3 ubiquitin ligases that promotes ubiquitination of many substrate proteins and controls numerous cellular processes. Skp1 is an adapter protein that binds directly to the F-box proteins. In this study, we have presented the first comprehensive analysis of the presence of peptides or proteins in the human pathogen Entamoeba histolytica having homology to Skp1protein. The occurrence of other protein components of the SCF complex has been identified from protein–protein interaction network of EhSkp1A. Studying the role of Skp1protein in this pathogen would help to understand its unique chromosome segregation and cell division which are different from higher eukaryotes. Further, owing to the development of resistance over several drugs that are currently available, there is a growing need for a novel drug against E. histolytica. Proteins from ubiquitin-proteasome pathway have received attention as potential drug targets in other parasites. We have identified four homologs of Skp1 protein in E. histolytica strain HM-1: IMSS. Molecular docking study between EhSkp1A and an F-box/WD domain-containing protein (EhFBXW) shows that the F-box domain in the N-terminal region of EhFBXW interacts with EhSkp1A. Therefore, the results of the present study shall provide a stable foundation for further research on the cell cycle regulation of E. histolytica and this will help researchers to develop new drugs against this parasite.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12639-022-01523-0.

Introduction

The ubiquitin–proteasome system (UPS) controls the degradation of several proteins and plays important roles in numerous cellular processes including cell cycle control (Reed 2006). Ubiquitination of protein is a multi-step process. The whole process is carried out by three enzymes, E1 ubiquitin‐activating enzyme that binds to the ubiquitin in an ATP-dependent manner and transfers it to the E2 conjugating enzyme, forming an E2 ubiquitin complex. The final step for the transfer of ubiquitin to substrate protein is mediated by E3 ligase. This is actually responsible for the attachment of the substrate protein and E2-ubiquitin complex. E3 complex finally binds to the substrate proteins that further leads to the degradation by 26S proteasome (Pickart 2001).

There are two distinct families of E3 enzymes: (a) Homologous to E6-AP Carboxyl Terminus (HECT) and (b) Really Interesting New Gene (RING). HECT E3s contain a N-terminal substrate-binding domain and a C-terminal HECT domain of approximately 350 amino acids. RING E3s contain a RING domain which is required to recruit E2 ~ ubiquitin and stimulate ubiquitin transfer. Both RING and HECT E3 ligases transfer ubiquitin to a lysine residue on the substrate (Deshaies and Joazeiro 2009; Rotin and Kumar 2009). Among the RING-type of E3 ligases, the SCF (Skp1, Cullins, F-box proteins) complex has been well studied. SCF complex consists of scaffold protein Cullin-1, E2-ubiquitin complex binding RING-box protein 1 and the adapter protein, S-phase kinase-associated protein 1 (Skp1) that presents the F-box protein to SCF complex (Zheng et al. 2002). Mammalian Skp1 is an important component of the SCF ubiquitin ligase complex, which promotes the ubiquitination of proteins involved in signal transduction and cell cycle progression (Bai et al. 1996; Xie et al. 2019). Skp1 in budding yeast is a kinetochore protein that can play an important role in cell cycle progression (Connelly and Hieter 1996). It is reported that fission yeast Skp1 plays an essential function in spindle morphology and segregation of nuclear membrane structures at anaphase. In yeast, the skp1 mutants show arch-like structures of anaphase spindles rather than straight ones (Lehmann and Toda 2004). Skp1 and Cul1 have been identified in purified centrosome in mammalian cells (Freed et al. 1999). O₂-dependent post-translational glycosylation of Skp1 promotes the formation of Skp1–Cullin-1–F-box protein complex in Dictyostelium (Sheikh et al. 2015).

F-box protein regulates substrates in various biological pathways and controls many aspects of cellular life including cell growth, cell division, cell signaling, cell survival and cell death (Cheng et al. 2019; Jandke et al. 2011; Jurado et al. 2008; Lutz et al. 2006; Noir et al. 2015). The F-box domain is a protein structural motif of approximately 50 amino acids. The F-box motif functions to perform protein–protein interaction. This motif is generally found in the amino-terminal region of proteins with other motifs in the carboxyl-terminal part; leucine-rich repeats (LLRs) and WD repeats being the two most common motifs. Depending on the nature of these motifs F-box proteins are divided into three subfamilies; FBXLs (a protein containing an F-box and LRRs), FBXWs (a protein containing an F-box and WD repeats), and FBXOs (a protein containing an F-box and other motifs) (Jin et al. 2004). Many SCF complexes present in yeast and human cells differ only in the F-box protein component.

In the present study, a Skp1 homolog, EhSkp1A has been identified and characterized from the human pathogen E. histolytica. It is a protozoan parasite responsible for amoebiasis and one of the top three parasitic causes of mortality (Kantor et al. 2018). Nitroimidazole derived compounds are generally recommended for treatment of amoebiasis but there is a growing need to develop new drugs against this human pathogen. During cell division and nuclear division, this parasite reveals some abnormal characteristics than other higher eukaryotes. Heterogeneity of DNA content has been observed in axenic cultures of E. histolytica trophozoites (Gangopadhyay et al. 1997). It has been observed from previous studies that, several checkpoint proteins that regulate the eukaryotic cell cycle are absent in this organism. Due to the absence of these checkpoint proteins, unregulated DNA synthesis and asymmetrical chromosome segregation may occur. Degradation of proteins in a regulated way has an important aspect in cell cycle regulation. Thus the substrate ubiquitination of the E3 enzymes plays a key role in several cellular processes, and the components of the SCF complex have emerged as potential target for drug development in future. Therefore as a component of the SCF E3 ubiquitin ligases, characterization of EhSkp1A would help to identify the unique cellular events in E. histolytica and also help to develop new drugs against this pathogen.

Materials and methods

Sequence retrieval and in silico analysis of EhSkp1A

Skp1 homologs in E. histolytica were retrieved by homology searches in NCBI (https://www.ncbi.nlm.nih.gov/) protein database. Multiple sequence alignment (MSA) and conserved domain analysis of these homologs were performed by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) (Sievers and Higgins 2018) and NCBI CDD (Conserved Domain Database) (https://www.ncbi.nlm.nih.gov/cdd/) search respectively. Sequence alignment of Skp1 protein was performed using the homologous Skp1 sequences of 12 different organisms i.e. Homo sapiens (Hs), Xenopus laevis (Xl), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Gallus gallus (Gg), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Plasmodium falciparum (Pf), Plasmodium malariae (Pm), Toxoplasma gondii (Tg), Leishmania donovani (Ld) and Entamoeba histolytica (Eh). Skp1 protein sequences from the representative eukaryotic organisms were collected from the UniProt database of protein sequences. Visualization of MSA was carried out using ESPript 3.0 program (https://espript.ibcp.fr/ESPript/ESPript/) (Gouet et al. 1999). Primary structural analysis of EhSkp1A was performed using the ProtParam tool (http://web.expasy.org/protparam) whereas secondary structural analysis was done through the SOPMA tool (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) (Geourjon and Deleage 1995) and PSIPRED protein structure prediction server (http://bioinf.cs.ucl.ac.uk/psipred/) (McGuffin et al. 2000). Protein–protein interactions and function analysis of EhSkp1A were performed using STRING v11 (functional protein association networks) (https://string-db.org/) (Szklarczyk et al. 2019) and PFP tool (an automated protein function prediction method that predicts Gene Ontology) (https://kiharalab.org/web/pfp.php) respectively (Khan et al. 2015).

Phylogenetic analysis

The amino acid sequences of E. histolytica Skp1 homologs and Skp1 protein sequences from different organisms having diverse origins were used to construct a phylogenetic tree using MEGA X software (Kumar et al. 2018). An unrooted phylogenetic tree was constructed using the Neighbour Joining (NJ) algorithm. Bootstrap analysis was performed with 1000 replications.

Homology modeling

3D structure of EhSkp1A was generated using its amino acid sequence employing SWISS-MODEL server (Guex and Peitsch 1997). GalaxyWEB server was used to generate the 3D structure of EhFBXW (Ko et al. 2012a). In this server Galaxy TBM method (Ko et al. 2012b) uses multiple templates for generating a reliable core structure. Unreliable loops or termini were re-built by using a refinement method, GalaxyRefine (Heo et al. 2013). The generated models were verified using PROCHECK (Laskowski et al. 1993), Verify3D programs (Luthy et al. 1992) available from the Structural Analysis and Verification Server (SAVES) (http://nihserver.mbi.ucla.edu/SAVES) and ProSA web server (Wiederstein and Sippl 2007). PROCHECK was used to check the stereochemical quality of protein structure, whereas the Verify3D program analyzed the compatibility of an atomic model (3D) with its own amino acid sequence (1D) to assess the 3D protein structure. ProSA was used to verify the 3D structures. Swiss-PdbViewer was used for energy minimization of the modeled protein structures (Guex and Peitsch 1997).

Ligand binding site prediction of EhSkp1A

The biochemical function of a protein mostly depends on its interaction with biomolecules such as ligands, other proteins or nucleic acids. Ligand binding site analysis of EhSkp1A was performed using GalaxySite web server (http://galaxy.seoklab.org/site) (Heo et al. 2014). This server predicts ligand binding site of a query protein by protein–ligand docking. Ligands used in this docking are predicted from the structures of template proteins with bound ligands.

Molecular docking study between EhSkp1A and EhFBXW

Molecular docking study between EhSkp1A and EhFBXW was performed using ClusPro web server (https://cluspro.bu.edu/publications.php) (Kozakov et al. 2017). The server conducts three computational steps to generate the result. The first step is to carry out rigid-body docking using PIPER, a docking program based on the Fast Fourier Transform correlation approach. Here billions of different random conformations are analyzed. The second step is to perform root-mean-square deviation (RMSD) based clustering of the 1000 lowest energy structures to find the largest cluster that may mimic the original structure and finally in step three, energy is minimized and the selected structures are refined. To analyze the binding affinity between EhSkp1A and EhFBXW, we have used the structure based method PRODIGY (PROtein binDIng enerGY prediction) (Xue et al. 2016). Interaction residues, interface area, and the number of non-bonded contacts were analyzed using the COCOMAPS (bioCOmplexes COntact MAPS) (Vangone et al. 2011) and PDBsum web server (Laskowski et al. 2018). In HawkDock server (Weng et al. 2019), MM/GBSA (Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation) free energy decomposition analysis was used to predict the binding free energy of the complex. Interface residues have been represented diagrammatically using the LigPlot program (Wallace et al. 1995).

Results

Identification and phylogenetic analysis of E. histolytica Skp1 homologs

From homology search, using the NCBI protein database we have identified four Skp1 homologs; EhSkp1A (NCBI Acc. No: XP_654128.1), EhSkp1B (NCBI Acc. No: XP_001913917.1), EhSkp1C (NCBI Acc. No: XP_651139.1), and EhSkp1D (NCBI Acc. No: XP_651670.1) in E. histolytica strain HM-1: IMSS. The human genome encodes only one Skp1 protein. Homolog of Skp1 from Homo sapiens (NCBI Acc. No: AAH20798.1) has been used as a query sequence. Conserved domain analysis showed that all Skp1 homologs of E. histolytica contain the BTB_POZ_SKP1 domain and Skp1 family dimerization domain. Sequence alignment of four Skp1 homologs from E. histolytica showed the variation of amino acid residues in F-box protein binding site. EhSkp1A and EhSkp1B have several conserved residues in F-box protein binding site. EhSkp1C and EhSkp1D showed comparatively lower sequence similarities with other EhSkp1 homologs found in E. histolytica (Fig. 1b, Supplementary Fig. 1). EhSkp1A, EhSkp1B, EhSkp1C and EhSkp1D shares 58.39%, 41.51%, 36.77% and 25.32% identity with the human Skp1 homolog respectively. As an essential component of SCF ubiquitin ligase complex, Skp1 protein in human mediates ubiquitination of several proteins which plays important role in many regulatory processes including signal transduction, cell cycle progression, and transcription. EhSkp1A shows high identity and similarity with human Skp1. Moreover, several conserved residues in F-box protein binding site present in EhSkp1A are similar to the human homolog (Fig. 1b). For this reason, we have selected EhSkp1A for further characterization to check whether it shows similar or different functions with the human homolog. The MSA of Skp1 protein sequences from 12 different organisms was carried out to study the evolutionary relatedness. MSA showed many variations in Skp1 protein sequences from different organisms. The observed sequence variations in Skp1 have been highlighted in white columns and the conserved one in red (Supplementary Fig. 2). Phylogenetic analysis showed that EhSkp1A and EhSkp1B are closely related to each other whereas EhSkp1C and EhSkp1D are grouped separately from other EhSkp1 homologs. Skp1 proteins from E. histolytica share low sequence similarities with the Skp1 protein homologs found in other protozoan parasites, therefore in phylogenetic tree, they clustered separately from other protozoan Skp1 homologs (Fig. 1a). Skp1 homologs in E. histolytica and other model organisms used in the study are given in Supplementary Table 6.

Fig. 1.

a Phylogenetic tree of Skp1 protein homologs. Organisms used in this study are Homo sapiens (Hs), Xenopus laevis (Xl), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Gallus gallus (Gg), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Plasmodium falciparum (Pf), Plasmodium malariae (Pm), Toxoplasma gondii (Tg), Leishmania donovani (Ld), Entamoeba histolytica (Eh). b Multiple sequence alignment of Skp1 protein homologs from 12 different organisms shows several conserved residues in F-box protein binding region (red box). Sequence variations have been highlighted in white columns and the conserved one in red (color figure online)

Structural analysis of EhSkp1A

The open reading frame (ORF) of EhSKP1A gene consists of 486 bp. ProtParam tool computed molecular weight, theoretical pI, aliphatic index, instability index, grand average of hydropathicity (GRAVY) and extinction coefficient at 280 nm of EhSkp1A protein to be 18,592.13, 4.79, 39.52, 80.56, − 0.428 and 12,615 (assuming all pairs of cysteine residues form cystines)/12490 (assuming all pairs of cysteine residues are reduced) M⁻¹ cm⁻¹ respectively. The value of the instability index (II) is lower than 50, indicating the stability of that protein. The lower GRAVY value indicates the hydrophilic nature of EhSkp1A, that means the protein makes better interaction with water. The high aliphatic index data gives an indication of protein stability over a wide temperature range. The amino acid sequence of EhSkp1A protein showed the presence of more negatively charged residues (Asp + Glu) than positively charged residues (Arg + Lys), which indicates the acidic nature of that protein. From conserved domain analysis, a BTB (Broad-Complex, Tramtrack and Bric a brac)/POZ (poxvirus and zinc finger) domain of 118 amino acid residues has been identified in the N-terminal region of EhSkp1A. Subcellular localization prediction of EhSkp1A predicted it to be a cytoplasmic protein. Secondary structure analysis showed that EhSkp1A consists of 63.58% alpha helix, 6.79% extended strand, 5.56% beta turn, and 24.07% random coil (Fig. 2a, c).

Fig. 2.

a Secondary structure prediction of EhSkp1A. b Secondary structure prediction of EhFBXW. c, d shows the pictorial representation of secondary structures of EhSkp1A and EhFBXW respectively

Functional analysis of EhSkp1A

The protein–protein interaction network plays an important role to analyze the biological function of a particular protein. STRING database (https://string-db.org) was used to find the functional partners of EhSkp1A. Probable functional partners of EhSkp1A were derived from the database of previous knowledge, high-throughput lab experiments, gene co-occurrence, gene co-expression, and textmining from other organisms. An interaction score > 0.7 (high confidence) was applied to construct the PPI networks. PPI network obtained from STRING consisted of 51 nodes and 569 edges. The average node degree and average local clustering coefficient were 22.3 and 0.776 respectively. PPI enrichment p value was < 1.0e−16. Proteins that physically interacts with EhSkp1A are Ring-box protein (EHI_151630), F-box/WD domain-containing protein (EHI_134350), ubiquitin-like protein nedd8 (EHI_103510), ubiquitin-conjugating enzyme family proteins (EHI_048700, EHI_003010, EHI_147470, EHI_178500), anaphase-promoting complex subunit 11(EHI_135100), cyclin family proteins (EHI_003890, EHI_121830), cullin-associated nedd8-dissociated protein 1(EHI_140650), cullin family protein (EHI_148160), ubiquitin-small subunit ribosomal protein s27ae (EHI_036530), suppressor of g2 allele of skp1(EHI_117820) and some hypothetical proteins (EHI_099230, EHI_139980, EHI_197130) (Fig. 3a). Most of the proteins identified as physical partners of EhSkp1A play essential roles in the ubiquitin-dependent protein catabolic processes. Among these, S-phase kinase-associated protein 1, Cullin family protein, F-box/WD domain-containing protein, and RING finger proteins are the important components of the SCF ubiquitin ligases. Co-expression analysis from other organisms showed that anaphase-promoting complex subunit 11 (EHI_135100) and cyclin family proteins (EHI_003890, EHI_121830) may be co-expressed as functional partners of EhSkp1A. Other functional partners of EhSkp1A identified from STRING analysis are summarized in Supplementary Table 3. Functional analysis of EhSkp1A has also been performed using the PFP server. PFP server gives the results based on PFP scores (scores denote; Very high confidence: > 20 K, High confidence: > 10 K, Moderate confidence: > 500, and Low confidence: ≥ 100). Selected GO-annotation terms (Molecular functions and Biological processes) are summarized in Supplementary Tables 1 and 2.

Fig. 3.

a Protein–protein interaction network of EhSkp1A. The edges indicate that the proteins are part of a physical complex. b Docked complex of EhSkp1A and EhFBXW, where EhSkp1A is depicted in red color and EhFBXW is depicted in green color. Interaction between EhSkp1A and EhFBXW shows that the F-box domain of EhFBXW interacts with EhSkp1A. c Hotspot residues at the interface in EhSkp1A (red circle). Dashed lines in brick red color indicate hydrophobic interactions and dashed lines in olive green color indicate hydrogen bonds (as obtained from the DIMPLOT module in LigPlot) (color figure online)

Structural and functional analysis of EhFBXW

EhFBXW consists of 423 amino acid residues with a theoretical molecular weight of 48,240.10 and a pI value of 6.33. A negative GRAVY value (− 0.260) indicates the hydrophilic nature of the protein. The value of instability index (II) is 36.69 (< 50), indicating the stable nature of the protein. Secondary structure prediction showed that EhFBXW consists of 23.40% alpha helix, 35.22% extended strand, 10.40% beta turn, and 30.97% random coil (Fig. 2b, d). Conserved domain analysis showed the presence of an F-box domain at the amino-terminal end and a WD40 repeat-containing domain in the carboxyl-terminal region. Interactors of EhFBXW have been identified from STRING database. Proteins that physically interact with EhFBXW are Skp1 family proteins (EHI_065310, EHI_118670, EHI_134960, EHI_066770), phosphoribulokinase/uridine kinase family proteins (EHI_051730, EHI_004610, EHI_196580), Ring-box protein 1(EHI_151630), cullin family proteins (EHI_061010, EHI_069300, EHI_118180, EHI_156450, EHI_167280, EHI_148160), ubiquitin-like protein nedd8 (EHI_103510), serine/threonine-protein kinase PLK1 (EHI_008410), ubiquitin-small subunit ribosomal protein s27ae (EHI_036530), anaphase-promoting complex subunit 11(EHI_135100), casein kinase 1(EHI_135100, EHI_005560, EHI_049390, EHI_151950, EHI_155240, EHI_156390), protein kinase domain-containing proteins (EHI_074780, EHI_121880, EHI_148220, EHI_000270) and some hypothetical proteins (EHI_065300, EHI_016470, EHI_043130, EHI_078420).

3D structure of EhSkp1A and EhFBXW

SWISS-MODEL server was used to carry out comparative homology modeling of EhSkp1A using 5k35.1.B (Skp1 from Homo sapiens) as a template. It is a fully automated server for comparative modeling of three-dimensional protein structures. The tertiary structure of EhFBXW was made using the GalaxyWEB server. 6NDU_A, 6M90_A 6M94_A, 6TTU_T (from Homo sapiens) were used as templates to generate the 3D structure of EhFBXW. Modeled 3D structures of the proteins were submitted to the Swiss-Pdb Viewer for energy minimization using the GROMOS96 43B1. 3D structure validations of EhSkp1A and EhFBXW have been summarized in Supplementary Table 4. Verify 3D results of model quality showed 80% of the residues have average 3D-1D score ≥  0.2. Ramachandran plot analysis revealed that maximum amino acid residues of modeled protein structures are within the allowed region, which indicates the presence of fewer steric clashes between atoms (Fig. 4a, b). Modeled structures showed good geometry because high-resolution structures generally tend to have better clustering within the allowed regions of the plot. Z-score values obtained from ProSA server were in the range of native proteins of similar size computed using NMR and X-ray methods, represented as black dots, and the plotted energy as a function of amino acid sequence position showed the energy remain negative for most of the amino acids which indicate the acceptability of the modeled structures (Fig. 4c–f). The predicted structural model of EhSkp1A has 8 helices, 3 strands, 1 beta bulge (antiparallel G1), 1 psi loop, 1 beta hairpin, 1 beta sheet, 12 helix-helix interactions, 10 beta turns, 1 gamma turn. The predicted structure of EhFBXW has 7 beta sheets (antiparallel), 17 beta hairpins, 17 beta bulges (8 antiparallel classic and 9 antiparallel G1), 25 strands, 10 helices, 16 helix-helix interactions, 50 beta turns and 10 gamma turns. The F-box motif of EhFBXW consists of three alpha helices. In the C-terminal region, tandem copies of WD-repeat fold together to form a circular protein domain (Fig. 5b).

Fig. 4.

Evaluation of the 3D structures of EhSkp1A and EhFBXW. a, b are the Ramachandran plot analysis of EhSkp1A and EhFBXW respectively. Residues in favoured regions (red), allowed regions (yellow), generously allowed regions (light yellow) and disallowed regions (white), c ProSA-web Z-score (highlighted as a black dot) of EhSkp1A and d EhFBXW, e ProSA-web plot of residue energies of EhSkp1A and f EhFBXW, where the window sizes of 40 and 10 residues are distinguished by dark and light green lines respectively. Positive values indicate problematic or erroneous parts of the structure (color figure online)

Fig. 5.

a Energy minimized structure of EhSkp1A predicted from SWISS-MODEL server. b Energy minimized structure of EhFBXW predicted from GalaxyWEB server (color figure online)

In silico analysis shows EhSkp1A interacts with the F-box domain of EhFBXW

From functional analysis (STRING), the various components of SCF complex have been identified as functional partners of EhSkp1A. There is a growing interest in knowing the function of F-box proteins and their association with the SCF complex because F-box proteins perform the vital role of delivering appropriate targets to the SCF complex. Using the N-terminal F-box motif, these proteins interact with the SCF complex by binding to the Skp1 subunit. So in this study, EhFBXW a homolog of F-box/WD domain-containing protein in E. histolytica, derived from the protein–protein interaction network of EhSkp1A, has been used for subsequent docking analysis. Energy minimized structures of EhSkp1A (Fig. 5a) and EhFBXW (Fig. 5b) were subjected to docking studies. ClusPro server performs docking calculations based on four different sets of energy parameters (balanced, electrostatic-favored, hydrophobic-favored, and van der Waals + electrostatics). The lowest energy score and the highest cluster member of four energy parameters are summarized in Supplementary Table 5. The energy calculated by PIPER is not directly related to binding affinity. Generally, low-energy regions show the tendency to generate large clusters of docked structures. The energy landscape ultimately determines the most likely conformation of the complex. Since no such previous information about the properties of the particular complex and the biophysical forces involved between them is available in E. histolytica, we have used balanced option to select the most likely model of the complex. This showed the highest member (149) in a cluster with a weighted score of − 1242 as center and − 1278.1 as lowest energy. This complex has been visualized by PyMOL. From molecular docking study, we have identified that the F-box domain of EhFBXW is responsible to interact with EhSkp1A (Fig. 3b). HawkDock server predicts that the binding free energy of the EhSkp1A-EhFBXW complex is − 148.93 kcal/mol. Binding affinity (predicted value of ΔG) between EhSkp1A and EhFBXW is − 14.3 kcal/mol. Negative values of ΔG indicate the stability of the protein complex. The interface area (Å2) of the complex is 1740.7. This interaction shows that the percentage of polar interface (63.76%) is greater than non-polar interface (36.24%). Analysis of this complex shows the abundance of the number of hydrophilic–hydrophobic interactions over the number of hydrophilic–hydrophilic interactions and hydrophobic–hydrophobic interactions. Figure 6a, b illustrate the binding free energy of each interface residue. Residues (hot spots at the interface in EhSkp1A) involved in the formation of hydrogen bonds, hydrophobic interactions, and other short contacts have been represented using the DIMPLOT module in LigPlot program (Fig. 3c). From the analysis of this complex, residues that identified as hotspots are 143 PHE, 125 ILE, 99 PHE, and 78 LYS for EhSkp1A, and 87 ARG, 88 SER, 76 THR 73 ILE and 57 GLN for EhFBXW. Hot spot residues of this interaction have been predicted based on their binding free energy and the number of residues with which a particular amino acid can interact at the surface of its interacting protein. Binding free energy of the hot spot residues at the interface of EhSkp1A-EhFBXW complex has been summarized in Table 1.

Fig. 6.

Interaction energy plots. a Binding free energy of interface residues in EhSkp1A. b Binding free energy of interface residues in EhFBXW

Table 1.

Binding free energy of the hot spot residues at the interface of EhSkp1A-EhFBXW complex

Binding free energy of hot spot residues at the interface in EhSkp1A		Binding free energy of hot spot residues at the interface in EhFBXW
Residues	Binding free energy (kcal/mol)	Residues	Binding free energy (kcal/mol)
B-PHE-143	− 5.16	A-THR-76	− 4
B-PHE-99	− 4.94	A-ARG-87	− 3.71
B-ILE-125	− 3.85	A-SER-88	− 3.66
B-LYS-78	− 2.81	A-ILE-73	− 2.89
		A-GLN-57	− 1.46

Ligand binding site of EhSkp1A

Ligand-binding site information of a protein provides valuable information for their functional studies and drug design. SXJ has been identified as a ligand of EhSkp1A ((13alpha, 18alpha)-2-cyano-3-hydroxy-12-oxooleana-2,9(11)-dien-28-oic acid). Arginine, Lysine, Isoleucine, Lysine, Aspartic Acid, Alanine, Threonine, and Glutamic Acid were identified as ligand-binding residues at 51, 53, 54, 57, 94, 96, 97, and 100 positions respectively. Interactions and predicted ligand binding sites have been analyzed using the LigPlot program (Supplementary Fig. 3).

Discussion

Characterization of proteins involved in cell cycle regulation would play an important role to understand the unique cell cycle events and design new drug against E. histolytica, where several abnormalities have been seen due to uncontrolled regulation of checkpoint proteins (Grewal and Lohia 2015). The present study has been undertaken to explore the role of a Skp1 homolog, in E. histolytica. Phylogenetic analysis of Skp1 homologs from diverse organisms shows the difference in sequence of EhSkp1A-D proteins compared to others. EhSkp1 homologs share lower sequence similarities with homologous sequences of Skp1 protein from other protozoan parasites like Plasmodium falciparum, Plasmodium malariae, Toxoplasma gondii, and Leishmania donovani. Physicochemical characterization of the amino acid sequence of EhSkp1A shows the hydrophilic nature of that protein. Structural analysis shows the abundance of more α-helices, which may play an important role to determine the global structure of EhSkp1A and its function. A BTB_POZ_SKP1 domain is present in the C-terminal region of EhSkp1A. The BTB domain is a protein–protein interaction motif that participates in many cellular functions, including targeting proteins for ubiquitination (Stogios et al. 2005). Several functional partners of EhSkp1A have been identified from STRING analysis. Proteins that show physical interaction with EhSkp1A have been analyzed. Most of these proteins have been identified as part of the SCF multi-subunit complex. Analysis of molecular function using GO-annotation also shows that the function of EhSkp1A is related to ubiquitin-protein ligase activity. From the interaction study and functional analysis, it can be deduced that EhSkp1A, as a component of the SCF complex may play an important role in ubiquitin-mediated protein degradation pathway. An F-box/WD domain-containing protein EhFBXW has been identified as a physical interactor of EhSkp1A. Since the F-box protein determines the substrate specificity within SCF complexes (Skaar et al. 2013), the characterization of EhFBXW may help to understand the overall regulation of the SCF complexes in E. histolytica. Here molecular docking study has been used to analyze the interaction between EhSkp1A and EhFBXW. Analysis of this interaction shows that the F-box domain in the N-terminal region of EhFBXW is responsible to interact with EhSkp1A. From the crystal structure of Skp1-Skp2-Cks1, it has been seen that human Skp1 interacts with N-terminal region of Skp2 (Hao et al. 2005). In Skp2 SCF complex, Skp2 targets several cell-cycle regulators and tumor suppressor proteins, including p27, for ubiquitin-mediated degradation (Fujita et al. 2008). However, in E. histolytica we didn’t find any homolog of Skp2 from sequence searching. In addition to EhSkp1 and the components of the SCF complex, the other proteins that have been identified through STRING analysis of EhFBXW include serine/threonine-protein kinase PLK1, that plays important functions throughout the M phase of the cell cycle, regulation of centrosome maturation, spindle assembly, regulation of mitotic exit and cytokinesis (Jackman et al. 2003; Jang et al. 2002; Lane and Nigg 1996); anaphase-promoting complex subunit 11, which is a component of the anaphase-promoting complex/cyclosome (APC/C) (Acquaviva and Pines 2006); casein kinase 1, that functions as regulators of signal transduction pathways (Schittek and Sinnberg 2014). F-Box/WD repeat-containing proteins have been seen to play an important role in cell cycle regulation by targeting several key regulators for ubiquitination and degradation (Zhang et al. 2020). Cdc4 is a WD-40 repeat F-box protein, a substrate recognition component of the SCF ubiquitin ligase complex in Saccharomyces cerevisiae that plays essential roles in cell cycle progression, signal transduction, and transcription (Kishi et al. 2008). β-transducin repeat-containing protein (β-TrCP) is a mammalian F-box protein of SCF ligase playing vital roles in cell cycle progression and signal transduction (Frescas and Pagano 2008). F-box and WD repeat domain containing 7 (FBXW7) is a tumor suppressor, targets oncoproteins for ubiquitination and degradation (Akhoondi et al. 2007). It has been found that the WD40 domain of FBXW7 binds to poly (ADP-ribose) (PAR) and expedites in early recruitment of FBXW7 to DNA damage sites for subsequent non homologous end-joining repair (Zhang et al. 2019).

Conclusion

This report shows the presence of four Skp1 homologs in E. histolytica genome. In silico characterization of a Skp1 homolog, EhSkp1A has been done in this study. 3D structures of EhSkp1A and its interactor EhFBXW have been predicted using homology-based approaches. Functional analysis shows that EhSkp1A may have an important role in ubiquitin-mediated protein degradation pathways. Several components of the SCF complex including cullin family protein, F-box/WD domain-containing protein, and RING finger protein have been identified from the protein–protein interaction network of EhSkp1A. Molecular docking study illustrates the direct interaction between EhSkp1A and the F-box domain of EhFBXW. In silico characterization of EhSkp1A and its interacting partners including the members of SCF E3 ubiquitin ligases delineate the role of EhSkp1A as well as SCF complexes in E. histolytica. This study would contribute to the further elucidation of events regulating unique cellular events in this early-branching protozoan. This fundamental study would help researchers to develop new drugs against this parasite.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors' contributions

RG and SD designed the study. RG performed most of the analysis. PB and SP assisted in molecular docking study. MD did the interaction energy plots. RG and SD wrote and edited the manuscript. All authors read and approved the final manuscript.

Data availability

Available on request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Animal rights

This article does not contain any studies with human participants or animals performed by the author.

Informed consent

Not applicable.

Ethical approval

The manuscript has been read and approved by all named authors. There are no other persons claiming for authorship. The order of authors listed in the manuscript has been approved by all of authors.