Ab initio modelling of an essential mammalian protein: Transcription Termination Factor 1 (TTF1)

Abstract Transcription Termination Factor 1 (TTF1) is an essential mammalian protein that regulates transcription, replication fork arrest, DNA damage repair, chromatin remodelling etc. TTF1 interacts with numerous cellular proteins to regulate various cellular phenomena which play a crucial role in maintaining normal cellular physiology, and dysregulation of this protein has been reported to induce oncogenic transformation of the cells. However, despite its key role in many cellular processes, the complete structure of human TTF1 has not been elucidated to date, neither experimentally nor computationally. Therefore, understanding the structure of human TTF1 is crucial for studying its functions and interactions with other cellular factors. The aim of this study was to construct the complete structure of human TTF1 protein, using molecular modelling approaches. Owing to the lack of suitable homologues in the Protein Data Bank (PDB), the complete structure of human TTF1 was constructed by ab initio modelling. The structural stability was determined with molecular dynamics (MD) simulations in explicit solvent, and trajectory analyses. The frequently occurring conformation of human TTF1 was selected by trajectory clustering, and the central residues of this conformation were determined by centrality analyses of the Residue Interaction Network (RIN) of TTF1. Two residue clusters, one in the oligomerization domain and other in the C-terminal domain, were found to be central to the structural stability of human TTF1. To the best of our knowledge, this study is the first to report the complete structure of this essential mammalian protein, and the results obtained herein will provide structural insights for future research including that in cancer biology and related studies. Communicated by Ramaswamy H. Sarma


Introduction
Ribosomes are essential cellular organelles, which are comprised of ribosomal proteins and ribosomal RNAs (rRNAs) that partake in protein synthesis in both prokaryotes and eukaryotes. They are encoded by ribosomal DNA (rDNA), distributed in clusters of $300-400 copies at both ends of the respective chromosomes (acrocentric chromosomes: 13, 14, 15, 21, and 22) that serves as the catalytic subunit of the protein translation machinery. These tandem repeats of rDNA copies create dense chromosomal regions called Nucleolar Organizer Regions (NORs), which consists of a non-transcribed spacer region flanked by pre-RNA coding regions and comprises 80% of the total RNA that is transcribed (Akamatsu & Kobayashi, 2015;Zhou et al., 2016). In mammalian cells, both the initiation and termination of rDNA transcription is mediated by a transcriptional regulator, which is an essential protein called Transcription Termination Factor 1 (TTF1), and the gene encoding this protein is located at 9q34.13 on the long arm of chromosome 9. TTF1 binds to DNA elements known as Sal box, which is located upstream and downstream of the rDNA gene repeats, consists of a SalI restriction site within the 11 bp consensus sequence, GGGTCGACCAG . Following its discovery as a transcriptional regulator, subsequent studies demonstrated that TTF1 is involved in polar replication fork arrest and also acts as an important chromatin remodelling factor (L€ angst et al., 1997;P€ utter & Grummt, 2002). Also, recent findings have demonstrated that TTF1 interacts with various DNA damage sensing proteins, including Cockayne Syndrome B (CSB) (Aamann et al., 2013), Mouse Double Minute 2 (MDM2) (Lessard et al., 2012), and tumour suppressor Alternative Reading Frame (ARF) (Lessard et al., 2010) protein. The mechanism and exact roles of TTF1 interacting with these proteins remain to be identified to date. Overexpression of TTF1 has been correlated in various tumours, indicating that TTF1 is required in higher quantities to meet the higher rate of ribosome biogenesis in tumour cells, owing to tumour hyperproliferation (Komatsu et al., 2016;Stults et al., 2009;Ueda et al., 2015). TTF1 is truly a multifunctional protein, and hence, it becomes important than ever to characterize the numerous unidentified roles of this protein in cellular physiology in both healthy and cancerous cells.
TTF1 has distinct functional domains, including an N-terminal regulatory domain (NRD), which is also responsible for the oligomerization of this protein (Sander & Grummt, 1997). Following oligomerization, TTF1 can loop the ends of rDNA together, creating a 'ribomotor' model by placing the promoter and terminator regions in proximity to efficiently recycle the transcription machinery (N emeth et al., 2008). Furthermore, TTF1 has a functional central domain and a C-terminal domain, which is essential for the activation and termination of Pol I-mediated transcription on a nucleosomal rDNA template (Boutin et al., 2019). The central domain has highly conserved DNA binding Myb/SANT-like domain which has a high degree of structural homology with the DNA binding domain of the Reb1 protein of Schizosaccharomyces pombe, and proto-oncoprotein c-Myb (Jaiswal et al., 2016;Park et al., 2018).
The only crystal structure of its yeast homolog protein, RNA Polymerase I enhancer binding protein (Reb1p) (Singh et al., 2010), bound to DNA, was solved to atomic resolution by our group (Jaiswal et al., 2016). The structure shows an N-terminal dimerization domain, a central DNA-binding domain, and the C-terminal transcriptional terminator domain. Using various mutants, it was demonstrated that mere binding of DNA to Reb1p is not sufficient for transcriptional termination. It was further shown that the interaction of the C-terminal domain of Reb1p with Replication Protein A (RPA) induces an allosteric change in the protein, which is necessary for ceasing the movement of RNA polymerase I (RNA Pol I), resulting in effective transcriptional termination. Also, the domain responsible for DNA binding was solved to atomic resolution and the residues involved in protein-DNA contacts were identified. This region consists of two Mybassociated domains (mybAD1 and mybAD2) and two Myb repeats (mybR1 and mybR2). The structure revealed that helices involved in this region make contacts with DNA at various residues (Jaiswal et al., 2016;Singh et al., 2010).
Owing to its role in various vital cellular functions, understanding the structure of TTF1 would provide important insights into the mechanistic aspect of the same. To date, there are no experimentally-determined structures or in silico models of TTF1 and hence our lab extremely is interested in solving the physical structure of this protein. So far, crystallization trials with purified protein have not proved to be successful, therefore we are attempting cryo-EM studies as well. Alternatively, computational modelling studies will provide a better understanding towards engineering this essential protein for future studies.
In the absence of experimentally-derived structures, homology modelling serves as a reliable method for the construction of protein structures. However, the reliability of the protein model depends on various factors, including the sequence identity between the template and target proteins. When the template-target identity falls below 30%, known as the twilight zone, the protein structure needs to be constructed by threading or ab initio methods (Khor et al., 2015). This is due to the fact that below the twilight zone, the evolutionary relatedness between the template and target is doubtful, and the confidence of the prediction is low (Chung & Subbiah, 1996). The worldwide experiment for protein structure prediction, Critical Assessment of protein Structure Prediction (CASP), ranked the iTasser (iterative threading assembly refinement) server as the best tool for ab initio protein modelling. In the latest CASP14 experiment conducted in 2020, the iTasser server (Zhang server) ranked the best among 47 groups (Roy et al., 2010;Yang et al., 2015). The iTasser server also ranked best in the previous CASP7, CASP8, CASP9, CASP10, CASP11, CASP12, and CASP13 experiments (Kryshtafovych et al., 2019). In the CASP9 experiments in 2010, the iTasser server was predicted to the best tool for protein function prediction. The iTasser server generates an atomic model of the submitted amino acid sequence by threading and iterative simulations (Yang et al., 2015). In this study, the structure of the TTF1 protein was constructed by molecular modelling using the iTasser server. The predicted models were validated and the structure was subjected to molecular dynamics (MD) simulations for 200 ns for studying the structural stability of TTF1 and determining the most frequently occurring stable conformation of this highly disordered protein. The aim of this study was to predict the complete structure of TTF1 and identify the residues that are crucial for stabilizing this conformation of the protein using computational modelling. The results of this study will prove to be important for understanding the structural, functional, and therapeutic role of this essential protein.

Sequence retrieval and sequence-based analyses
The sequence of TTF1 was retrieved from UniProtKB (UniProtKB accession number: Q15361). The physicochemical properties of TTF1 were analysed using ProtParam (Gasteiger et al., 2005), and the disorder profile was analysed using DisoPred version 3.1 (Jones & Cozzetto, 2015;Ward et al., 2004).

Ab initio modelling of TTF1
The structural homologues of human TTF1 in the PDB was searched using BLASTp and threading-based approaches, for identifying suitable templates for homology modelling. Due to the lack of suitable structural templates, the structure of human TTF1 was modelled using ab initio modelling, using the iTasser server (Roy et al., 2010). In the iTasser algorithm, the final models are selected using the SPICKER program for clustering the generated structures. The structure of TTF1 generated by iTasser was initially minimised using the Yasara energy minimization server, with the Yasara force field (Krieger et al., 2009). The energy minimised structure was then validated using Ramachandran plot analysis and ProSA (Laskowski et al., 1993;Wiederstein & Sippl, 2007).

Functional validation of TTF1 constructed by ab initio modelling
The models generated by iTasser were functionally validated using the TM-align program for determining the structures in the PDB that are structurally, and thus functionally, similar to the models of TTF1 constructed by ab initio modelling. This program determines the similarity between proteins on the basis of the TM-score, a scoring function that provides a quantitative measure of topological similarity between proteins (Zhang & Skolnick, 2005). The values > 0.5 indicates the models to be of correct topology (Zhang & Skolnick, 2004). The models were further validated using the COACH and COFACTOR programs for predicting the ligand binding sites, based on the similarity of the protein folds with functional templates (Yang et al., 2013;Zhang et al., 2017). The result of ligand binding site prediction was mapped to the results of sequence-based conserved domain (CD) analyses using the CD search tool of NCBI (Lu et al., 2020). The molecular function of the modelled protein was further validated by consensus-based gene ontology (GO) search.

MD simulations
The model of TTF1 obtained by ab initio modelling was subjected to MD simulations for 200 ns using Flare v4, which is based on the OpenMM Toolkit, for studying the structural stability and determining any possible conformational changes of TTF1. The protein was then prepared in Flare v4 at pH 7.4, and solvated in transferable intermolecular potential 4-point (TIP4P) solvent using a buffer of 10 Å thickness. The system was subsequently neutralised by the addition of 28 Clions. The system was then minimized until the energy tolerance reached 0.25 Kcal/mol, and subsequently equilibrated for 200 ps. It was then finally subjected to 200 ns MD simulations at a temperature of 298 K and a pressure of 1 bar, using the XED force field and the NPT ensemble. The timestep was set to 2 fs. The values of root mean square deviation (RMSD), root mean square fluctuations (RMSF), and radius of gyration (RoG) of the protein backbone throughout the trajectory was analysed using the vmdICE plugin in VMD v1.9.3 (Humphrey et al., 1996;Knapp et al., 2010). The occupancy of the interresidue hydrogen bonds throughout the 200 ns trajectory and in the last 50 ns was determined using VMD v1.9.3. The portion of the trajectory following equilibration was clustered using Chimera v1.14, and the representative frame of the most populated cluster was selected as the most frequently occurring conformation of TTF1 (Pettersen et al., 2004). MD simulations were also performed in triplicate using BIOVIA Discovery Studio Client, with the same simulation parameters, for 200 ps (Cava et al., 2021).

Sequence-based analyses
The results of sequence-based analysis with ProtParam showed that TTF1 is an unstable hydrophilic protein, as revealed by an instability index of 51.13 and grand average of hydrophobicity (GRAVY) of À0.939. This was corroborated by the results of disorder prediction, which showed that more than 50% of the residues of TTF1 are disordered ( Figure 1). The results of disorder prediction further revealed that residues 1-3, 689-696, 700-701, 709, and 903-905 were disordered and had protein binding properties ( Figure S1). The physicochemical properties predicted by ProtParam are enlisted in Table 1.

Ab initio modelling and structural validation of TTF1
The results of template search using BLASTp against the PDB revealed that the highest target-template coverage was 4%, which was well below the twilight zone for homology modelling (Rost, 1999). Therefore, the structure of human TTF1 could not be modelled using the template-based methods in comparative modelling. The complete structure of human TTF1 was therefore modelled using ab initio methods, using the iTasser server. The confidence of the models predicted by iTasser are indicated by the confidence score (C-score), which measures the quality of the models generated by iTasser. The C-scores range between À5 and 2, with higher values indicating predictions of higher confidence, while lower values of C-score indicate predictions of lower confidence (Roy et al., 2010). In this study, the model with the highest C-score of À0.60 was selected for subsequent analyses. This model was further minimised using Yasara, and the energy minimised structure was validated using ProSA (Krieger et al., 2009;Wiederstein & Sippl, 2007). The results of ProSA validation revealed that the model of TTF1 was comparable to those of structures of similar size in the PDB, which had been determined using X-ray crystallography ( Figure 2A). Analysis of the Ramachandran plot with ProCheck revealed that only 1.0% of the residues were in the disallowed regions of the plot, while 82.9% and 14.1% of the residues were in the most favoured and additional allowed regions, respectively ( Figure 2B).

Functional validation of TTF1
The results of analysis with TM-align revealed that the model of TTF1 generated by iTasser (Figure 3) was structurally most similar to cas13b (PDB ID: 6AAY), which is an RNA-binding protein with RNase activity from a gram negative bacterium Bergeyella zoohelcum (Zhang et al., 2018). The human TTF1 protein is a DNA-binding protein that plays an important role in transcriptional termination. The TM-score of the alignment was 0.960, indicating correct topology, and the RMSD between the generated model of TTF1 and cas13b was 2.29 Å, indicating high structural similarity between the two functionally very dissimilar proteins. The structural similarity between TTF1 and cas13b indicated that the model of TTF1 obtained herein, possesses potential nucleic acid binding properties, similar to cas13b. The results of ligand binding analyses with COFACTOR and COACH revealed that residues 620-626 of the TTF1 model have potential binding property to the ligand phosphoaminophosphonic acid-adenylate ester (ANP). ANP is a non-hydrolysable analogue of ATP, and comprises triphosphate, adenine, and ribose sugar moieties, and has a composition similar to that of DNA. This indicated that the model of TTF1 predicted using iTasser has potential nucleic acid binding properties, and logically relates to the DNAbinding properties of TTF1 reported in literature and also been validated in our lab using the purified TTF1 protein  . The ligand binding properties of the model of TTF1 were predicted to be most similar to those of the recombinase A protein of Escherichia coli (PDB ID: 3CMV), which possesses single-stranded DNA binding properties. These results indicated that the model of TTF1 possesses potential DNA binding properties, in agreement with the reports in existing literature and our experimental data (not shown here). The results of CD analyses revealed that residues 621-677 of TTF1 comprises the Myb-like DNA binding domain of TTF1 (pfam accession number: 13921) ( Figure S2). The results of sequence-based CD analysis of TTF1 corroborated with the results of structure-based ligand binding site prediction by COFACTOR and COACH, further validating the DNA-binding potential, and thus the functional validation of the model constructed using iTasser. Furthermore, the residues with ANP-binding properties mapped to the DNA-binding domain of TTF1, confirming nucleotide binding properties of the structure generated by iTasser.
The results of consensus-based GO prediction revealed that the molecular function of the TTF1 protein model was associated with GO terms GO:0035639 (purine ribonucleoside triphosphate binding), GO:0032559 (adenyl ribonucleotide binding), and GO:0043167 (ion binding), with GO scores of 0.40, 0.40, and 0.39, respectively. These results further confirm the nucleotide binding properties of the structure obtained with iTasser. The results of functional validation thus strongly  implies that the TTF1 model obtained using ab initio modelling has potential nucleic acid-binding properties, which is in agreement with the data reported in literature and experimentally observed in our lab.

Trajectory analyses
The structural model of TTF1 obtained by ab initio modelling was subjected to 200 ns MD simulations for investigating the structural stability and determining any possible conformational changes in TTF1. Trajectory visualization revealed that the protein stabilised after 20 ns and remained stable thereafter. This was further observed in the values of root mean square deviation (RMSD), which became steady after 20 ns ( Figure 4). As depicted in the Figure 4A, the values of RMSD became increasingly steady after 100 ns, and remained steady thereafter, with fluctuations in the RMSD values being in the range of 1-1.5 Å. This indicated that the system had reached equilibrium after 100 ns and remained stable thereafter. This was further corroborated by the values of RoG ( Figure 4B), which remained steady after 100 ns. The RoG is an indicator of structural compactness, and fluctuations in the values of RoG indicates protein unfolding. The fact that the values of RoG became steady after 100 ns indicated that the system was stable and compact during the production run. Analysis of the values of RMSF revealed that some residues had higher flexibility, as indicated by the RMSF values, which were higher than 1.5 Å. The higher flexibility of these residues could be attributed to the fact that these residues mapped to the disordered regions predicted using DisoPred (Figures 1 and 4C). As mention in the method section, we also did simulation of 200 ps in triplicate ( Figure S6

Determination of frequently occurring conformation of TTF1
It has been reported that intrinsically disordered proteins can have more than one stable conformation, which aid in performing diverse cellular functions. Since TTF1 is a highly disordered protein, it is most likely to exist in more than one conformation. Therefore, the simulation trajectory was clustered using Chimera v1.14 for identifying the most frequently occurring conformation. Hence, the frame of the most populated cluster was selected to represent the same ( Figure 5A, for the coordinate file refer to the supplementary section). The oligomerization domain (residues 1-320), Myb domain 1 (residues 612-660), Myb domain 2 (residues 661-745), and chromatin remodelling region (residues 323-445,) (L€ angst et al., 1997;Park et al., 2018;Sander & Grummt, 1997) were mapped to the complete structure of TTF1 obtained herein as shown in Figure 5B.

Centrality analyses
In order to identify the residues that are important for stabilizing the most frequently occurring conformation of TTF1, the RIN of the frequently occurring conformation of TTF1 was determined using Cytoscape v3.8.2 (Figure 6), and the central residues were identified using the RINspector plugin, based on the RCA Z-scores. In the RIN, the nodes indicate the residues, while the edges represent the intra-residue interactions. Residues with RCA Z-scores ! 2 were considered to be central to the structural stability of the protein.
As depicted in Figure 6, the residues with Z-scores ! 2 are coloured in yellow, and those with Z-scores ! 4 are represented in red. The bigger nodes indicate residues with higher values of Z-scores. The RIN revealed two interaction clusters, with one cluster being located in the oligomerization domain of TTF1, and the other being located towards the C-terminal region of the protein ( Figure 6A and B). The Z-scores of the residues in the interaction cluster in the oligomerization domain were higher than those of the residues in the C-terminal domain, indicating that the interaction cluster in the oligomerization domain plays a more crucial role in the stability of the human TTF1 protein than that of the interaction cluster in the C-terminal domain. The Z-scores of the central residues determined by centrality analysis are enlisted in Table 2.

Intra-residue hydrogen bonds
Hydrogen bonds with occupancy ! 75% and ! 85% throughout the 200 ns trajectory and in the last 50 ns, respectively, were considered to be important for the structural stability of the protein (occupancy is provided in Table S1). The frequency of the hydrogen bonds throughout the trajectory and in the last 50 ns was determined using VMD. The occupancy of the intra-residue hydrogen bonds formed by the central residues is provided in Table S1. The results of interaction analyses revealed that residues K17, E27, Q30, E35, R164, W198, and N228 of the oligomerization domain, K434 of the chromatin remodelling region, and F657 of the myb/SANT-like-1 domain were most crucial to the structural stability of TTF1 for the above confirmation, as indicated by the number of intra-residue hydrogen bonds and the occupancy of the hydrogen bonds throughout the trajectory.

Discussion
TTF1 is a crucial multifunctional nucleolar protein that regulates both transcription initiation as well as termination of the ribosomal genes by binding to specific motif sequence and also arrests replication fork in polar fashion (Akamatsu & Kobayashi, 2015). In addition, TTF1 regulates the transcription of genes transcribed by RNA polymerase I as well. Despite its major regulatory role in mammalian transcription, replication and chromatin remodelling, the complete structure of human TTF1 remains to be elucidated to date. A partial structure of human TTF1 has been predicted by AlphaFold v2.0, which uses artificial intelligence for predicting the 3-dimensional structure of proteins (AlphaFold AF-Q15361-F1). However, the structure predicted by AlphaFold is partial (residues 491-866), and the remaining residues are largely unfolded, with very low confidence of prediction (Jumper et al., 2021). In order to determine the stability of the TTF1 model constructed herein, the model of TTF1 from AlphaFold was also simulated for 200 ps for comparative analysis with our predicted model of TTF1. Trajectory analysis revealed that our predicted structure of TTF1 is more stable than the predicted structure of AlphaFold, as revealed by the comparative RMSD and RMSF plots (as shown in Figure S7 and S8). The RMSF values of some of the residues of the AlphaFold structure were very high, being in the order of 10-12 Å. Also, we have provided a superimposition of TTF1 modelled by our method and AlphaFold of residue 491 À 866 (as mentioned above) in Figure S8 for better comparison (with energetics). Since TTF1 is a multidomain protein, the various domains are important for its diverse biological functions. Hence, it becomes important to know the structure of this essential protein in it's entirely. In this study, we therefore attempted to construct the complete structure of the human TTF1 protein using ab initio modelling and MD simulations. We identified the residues that are central to the structural stability of human TTF1 by network analyses. The structural model of TTF1 was validated by Ramachandran plot analysis, which revealed that only 1.0% of its residues were in the Figure 6. The central residues of TTF1 identified by RIN and centrality analyses in the A) 3-dimensional structure of TTF1. B) The central residues in the RIN of TTF1 in 2 D representation. The nodes and edges represent the residues and inter-residue interactions, respectively. The size of the nodes corresponds to the value of the RCA Z-score, with bigger nodes corresponding to residues with higher values of Z-scores. The residues with RCA Z-score ! 2 and ! 4 are indicated in yellow and red, respectively. disallowed regions, indicating highly satisfactory quality of the proposed model. To the best of our knowledge, this study is the first to report the complete structure based on most frequently occurring conformation of human TTF1 protein. There are several webservers for protein structure prediction, including SwissModel, Rosetta, etc., in addition to standalone software like Modelller. However, the structure of human TTF1 could not be modelled using these tools, owing to the lack of suitable structural templates with sequence coverage above the twilight zone, which resulted in poor confidence scores or partial models (like that of AlphaFold). The structure of human TTF1 was therefore modelled using the ab initio modelling approach of iTasser. The iTasser webserver is an integrated platform for ab initio modelling. The model of TTF1 thus obtained from iTasser was subjected to functional validation and GO analysis for establishing the functional relevance. MD simulations are frequently used for obtaining atomic level insights into the structural dynamics and behaviour of biomolecules. The stability of the model was subsequently evaluated by MD simulation for 200 ns and further triplicate runs were performed at 200 ps to validate the same and the trajectory was analysed for investigating structural stability and hydrogen bond frequency. The most frequently occurring conformation of the human TTF1 protein was obtained by trajectory clustering, and the residues that play a central role in the structural stability of TTF1 were identified by network analysis and by determining their residue centrality. The results of RIN analysis and computation of centrality measures revealed two interaction clusters in the structure of human TTF1, with one in the oligomerization domain of TTF1 and the other in the C-terminal domain which is in accordance with previous published works.
Despite the presence of an oligomerization domain, there is no information regarding the oligomerization state (how many homomers involved) of TTF1. Using truncated human and murine TTF1 proteins, Evers and Grummt first reported species-specific sequence differences in the DNA-binding region of mammalian TTF1 . The tryptophan residue at 688 position replaced by lysine residue leads to inability in binding of murine TTF1 to DNA showing functional and structural significance of Myb sequence conservation . Our predicted structure is in agreement to the above experimental outcome and also with physical structure data (solved to 1.5 A 0 ) of its yeast homolog protein Reb1 (Jaiswal et al., 2016), showing involvement of Myb repeats in stabilizing and binding of DNA ( Figure 3 and Table 2). The data further indicated that the residue cluster in the oligomerization domain plays a more significant role in the stability of TTF1, compared to that in the C-terminal domain. Experimentally the N-terminal oligomerization domain has been shown to play important regulatory function (Akamatsu & Kobayashi, 2015) while the C-terminal domain is involved in transcription termination (L€ angst et al., 1997). The data obtained herein could not be compared with information from experimental studies, due to the fact that there are no reports on the structural attributes of TTF1 to date and also there is lack of structural homologues (mammalian/human) of TTF1 in the PDB, which makes this study the first of its kind for this essential mammalian protein. The only closest homology we found it shares is with a bacterial RNA binding protein (as mentioned in result) which is entirely a functionally dissimilar protein. Our lab is actively engaged in experimental studies to obtain critical insights into the structural and functional characteristics of TTF1. In the absence of experimentally-derived structural data pertaining to the human TTF1 protein, we believe that the results of our study provide valuable structural information, including domain architecture, and their characteristics. Hence, our study could facilitate future studies aimed towards understanding the mechanism underlying the function of the human TTF1, including its interaction with other protein, and for engineering this protein with the purpose of solving its physical structure, drug design and therapeutic applications.

Conclusion
Conclusively, this is very first study to report the complete structure based on most frequently occurring stable conformation of the essential human protein TTF1 using computational modelling, and identify the residues and its characteristics that are central to the structural stability of -2.069 Helix the protein. The predicted complete structure provides essential information regarding the residue clusters, and can aid in understanding the mechanism underlying the activation of other genes and in expression studies both in healthy and cancerous cells. The residues that are crucial for the structural stability of TTF1 can be mutated for investigating their role in different disease conditions. However, subsequent computational and experimental studies are necessary for obtaining deeper structural insights of this protein.