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Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach

View Article: PubMed Central - PubMed

ABSTRACT

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

No MeSH data available.


Number of intermolecular hydrogen bonds formed between the protein and DNA in the native and mutant complexes.Color scheme: native (black), R131W (red), R131Q (green), and R203C (blue).The R203C mutant complex formed the least number of intermolecular hydrogen bonds throughout the simulation period compared to the native complex, followed by the R131Q mutant complex. The R131W mutant complex formed less number of intermolecular hydrogen bonds than the native complex but more than the R131Q and R203C mutant complexes.
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pone.0174953.g005: Number of intermolecular hydrogen bonds formed between the protein and DNA in the native and mutant complexes.Color scheme: native (black), R131W (red), R131Q (green), and R203C (blue).The R203C mutant complex formed the least number of intermolecular hydrogen bonds throughout the simulation period compared to the native complex, followed by the R131Q mutant complex. The R131W mutant complex formed less number of intermolecular hydrogen bonds than the native complex but more than the R131Q and R203C mutant complexes.

Mentions: Triplicate runs of 50ns MD simulations using Gromacs were performed to understand the stability, functionality, and folding variations between the native and mutant complexes at an atomic level. Trajectory files obtained from the triplicate runs of the native complex were utilized to construct a Block-based root-mean-square deviation (RMSD) plot. This analysis allows us to compare and identify the most highly converged trajectory of the native complex for further analysis (Fig 3). Among the three runs, the native complex marked in violet (run 2) showed better convergence pattern (Fig 3). Runs 1 & 3 exhibited greater deviation pattern and lower convergence in the Block-based RMSD plots, which were used to plot the RMSD graph illustrated in S3A and S3B Fig. Trajectory files from run 2 of the native and mutant complexes were utilized to measure the impact of the mutations on protein-DNA interactions using various Gromacs utilities. The RMSD analysis was used to measure the changes in the deviation pattern between the native and mutation complexes (Fig 4). The native complex exhibited a deviation between ~0.2nm and ~0.4 nm until 30ns, and convergence was finally observed at the end of 50 ns, with an RMSD value of ~0.3nm (Fig 4). The R203C mutant complex exhibited the highest deviation at 10ns upto an RMSD value of ~0.5nm, which later converged to ~0.35nm at 20ns. An increase in the RMSD value up to ~0.45nm was observed at the end of the 50ns simulation. Meanwhile, the R131W mutant complex and native complex exhibited similar deviation patterns. The R131Q mutant complex exhibited a deviation between ~0.25 and ~0.35 that converged at 50ns, with an RMSD value of ~0.3nm (Fig 4). A root mean square fluctuation (RMSF) plot was constructed to measure the observed differences in fluctuation pattern between the native and mutant complexes. Overall, a greater fluctuation pattern was observed at terminal residues of the native and mutant complexes. We observed greater fluctuations in the R203C mutant complex (up to ~0.30nm), followed by the R131W and R131Q mutant complexes, which exhibited fluctuations of up to ~0.27nm. However, the native complex exhibited a minimal fluctuation of upto ~0.20nm (S4 Fig). The observed differences in the RMSD (Fig 4) were further validated by assessing the number of intermolecular hydrogen bonds formation between the protein and DNA in the native and mutant complexes (Fig 5). The average number of intermolecular hydrogen bonds formed between the native protein and DNA was 31.11. In mutant complexes, R203C showed the least number of intermolecular hydrogen bonds, with an average number of 20.075, whereas R131Q and R131W showed average numbers of intermolecular hydrogen bonds of 36.05 and 34.667, respectively (Fig 5). The least number of hydrogen bonds formation in the R203C mutant complex correlates with the higher deviation pattern observed in the RMSD analysis. The larger deviation explains the destabilizing effect because fewer hydrogen bonds were formed.


Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach
Number of intermolecular hydrogen bonds formed between the protein and DNA in the native and mutant complexes.Color scheme: native (black), R131W (red), R131Q (green), and R203C (blue).The R203C mutant complex formed the least number of intermolecular hydrogen bonds throughout the simulation period compared to the native complex, followed by the R131Q mutant complex. The R131W mutant complex formed less number of intermolecular hydrogen bonds than the native complex but more than the R131Q and R203C mutant complexes.
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pone.0174953.g005: Number of intermolecular hydrogen bonds formed between the protein and DNA in the native and mutant complexes.Color scheme: native (black), R131W (red), R131Q (green), and R203C (blue).The R203C mutant complex formed the least number of intermolecular hydrogen bonds throughout the simulation period compared to the native complex, followed by the R131Q mutant complex. The R131W mutant complex formed less number of intermolecular hydrogen bonds than the native complex but more than the R131Q and R203C mutant complexes.
Mentions: Triplicate runs of 50ns MD simulations using Gromacs were performed to understand the stability, functionality, and folding variations between the native and mutant complexes at an atomic level. Trajectory files obtained from the triplicate runs of the native complex were utilized to construct a Block-based root-mean-square deviation (RMSD) plot. This analysis allows us to compare and identify the most highly converged trajectory of the native complex for further analysis (Fig 3). Among the three runs, the native complex marked in violet (run 2) showed better convergence pattern (Fig 3). Runs 1 & 3 exhibited greater deviation pattern and lower convergence in the Block-based RMSD plots, which were used to plot the RMSD graph illustrated in S3A and S3B Fig. Trajectory files from run 2 of the native and mutant complexes were utilized to measure the impact of the mutations on protein-DNA interactions using various Gromacs utilities. The RMSD analysis was used to measure the changes in the deviation pattern between the native and mutation complexes (Fig 4). The native complex exhibited a deviation between ~0.2nm and ~0.4 nm until 30ns, and convergence was finally observed at the end of 50 ns, with an RMSD value of ~0.3nm (Fig 4). The R203C mutant complex exhibited the highest deviation at 10ns upto an RMSD value of ~0.5nm, which later converged to ~0.35nm at 20ns. An increase in the RMSD value up to ~0.45nm was observed at the end of the 50ns simulation. Meanwhile, the R131W mutant complex and native complex exhibited similar deviation patterns. The R131Q mutant complex exhibited a deviation between ~0.25 and ~0.35 that converged at 50ns, with an RMSD value of ~0.3nm (Fig 4). A root mean square fluctuation (RMSF) plot was constructed to measure the observed differences in fluctuation pattern between the native and mutant complexes. Overall, a greater fluctuation pattern was observed at terminal residues of the native and mutant complexes. We observed greater fluctuations in the R203C mutant complex (up to ~0.30nm), followed by the R131W and R131Q mutant complexes, which exhibited fluctuations of up to ~0.27nm. However, the native complex exhibited a minimal fluctuation of upto ~0.20nm (S4 Fig). The observed differences in the RMSD (Fig 4) were further validated by assessing the number of intermolecular hydrogen bonds formation between the protein and DNA in the native and mutant complexes (Fig 5). The average number of intermolecular hydrogen bonds formed between the native protein and DNA was 31.11. In mutant complexes, R203C showed the least number of intermolecular hydrogen bonds, with an average number of 20.075, whereas R131Q and R131W showed average numbers of intermolecular hydrogen bonds of 36.05 and 34.667, respectively (Fig 5). The least number of hydrogen bonds formation in the R203C mutant complex correlates with the higher deviation pattern observed in the RMSD analysis. The larger deviation explains the destabilizing effect because fewer hydrogen bonds were formed.

View Article: PubMed Central - PubMed

ABSTRACT

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

No MeSH data available.