Mutation screeningA total of 177 single nucleotide polymorphisms (SNPs) obtained from the various databases were located within amino acid residues 1-451 of the αIIb subunit corresponding to the β-propeller domain (Table S1).Pathogenicity analysisAll identified SNPs were further analyzed for their pathogenicity using PredictSNP17, out of which sixty were predicted to be deleterious and pathogenic using tools, such as PredictSNP, MAPP, PhD-SNP, Polyphen-1, Polyphen-2, SIFT, and SNAP (Table S2). Not surprisingly, most of these mutations that were associated with specific functional defects correlated with GT type 1.Stability analysisMutations affecting the structural stability of a protein are often responsible for the disease phenotype. Hence, mutations with deleterious and pathogenic consequences were studied further. These mutations were analyzed for their impact on structural stability using the iStable18 tool, which is used for analyzing the changes in stability as a result of the mutations. Twenty-seven mutations were found to destabilize the structure of integrin αIIbβ3 based on the DDG scores obtained using tools, like MuPro, iStable and I-Mutant (Table S3).Conservation analysisAmino acid residues that are highly conserved during the course of evolution are unlikely to be mutated. Accordingly, conservation analysis was done for all the identified mutations and only those that involved highly conserved residues were studied further. Nine mutations had occurred in highly conserved regions as indicated (Fig. 2A). Among these, while residues G44, E355, and G401 are highly conserved and exposed on the β-propeller surface, residues P176, G267, G296, and G321 although highly conserved, are buried. The other residues, namely F320 and L343 are also conserved and buried (Fig. 2B). Although these mutations were reported to be functionally important, structural changes caused by them have not been studied in detail.Figure 2Evolutionary conservation analysis of the αIIb amino acid sequence (A) with the highly conserved amino acid residues depicted on the β-propeller structure (B).Molecular docking studiesSome of the prominent and functionally significant mutations reported in the literature were chosen for this study (Table 1). The wild-type, G296R, F320S, G321W, E355K, and G401C αIIbβ3 structures were subjected to molecular docking analysis, which revealed the binding energy, docking score, and binding orientation between the αIIb and β3 subunits (Table 2). In general, mutant structures with lower binding energies were found to form highly stable complexes with greater binding affinity. Among the five mutations, E355K and G401C had the highest binding energies indicating that these two mutations might affect fibrinogen binding to mutant αIIbβ3 complexes when compared to the wild-type due to changes in the interactions (Fig. 3A-C). Therefore, E355K and G401C mutations were considered for MD simulations to better understand their deleterious consequences as detailed below.Table 1 Prominent β-propeller mutations causing Glanzmann thrombasthenia.Table 2 Docking scores of wild-type, G296R, F320S, G321W, E355K, and G401C αIIbβ3 structures.Figure 3Three dimensional interactions of αIIbβ3 complexes after docking with fibrinogen. Wild-type (A), E355K (B), and G401C (C). Chains A and B represent aIIb and b3 subunits, respectively.MD trajectory analysisE355K compromised stability as compared to G401CRMSD was calculated to determine the convergence and deviations with time based on Cα atoms in the wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen (Fig. 4A). The average RMSD values of E355K and G401C αIIbβ3 complexes with fibrinogen were observed to be higher as compared to that of the wild-type (Table 3). The protein structures were stabilized with fixed values which indicated that the docked complexes of E355K and G401C had deviations throughout the simulation period (500 ns), with E355K recording the highest fluctuation. Similarly, RMSF was calculated which indicated the changes in flexibility of the wild-type, E355K, and G401C αIIbβ3 structures over time when bound with fibrinogen. The E355K structure displayed more flexibility implying stability changes when compared to the wild-type, with the G401C structure showing slightly less fluctuation (Fig. 4B). The average RMSF values of E355K and G401C αIIbβ3 complexes with fibrinogen were observed to be higher as compared to that of the wild-type (Table 3).Figure 4RMSD and RMSF plots of wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen following MD simulations (500 ns). (A) RMSD values corresponding to the fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes. The x-axis represents time in ns, while the y-axis represents RMSD values in nm. (B) Graphical representation of RMSF values corresponding to the fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes. The x-axis indicates amino acid residues, while the y-axis indicates RMSF values in nm.Table 3 Average RMSD, RMSF, Rg, and SASA values of fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes.The Rg value of a protein structure is used to calculate the distribution of atoms from the centre of the mass, which denotes the compactness of the protein structure. The competence and folding of the wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen were observed at different time points during the trajectory, which revealed that when compared to the wild-type fibrinogen-bound αIIbβ3 complex, both E355K and G401C complexes exhibited more deviations thereby compromising the compactness of the protein structure (Fig. 5A). The average Rg values of fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes were 3.83, 4.31, and 4.11 nm, respectively (Table 3). SASA was performed in order to identify the changes caused by mutations in the hydrophobic core of the protein structure, which showed changes in SASA associated with both fibrinogen-bound E355K and G401C complexes as compared to the wild-type (Fig. 5B). The average SASA values of fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes were 673.33 +/- 29.10, 746.03 +/- 15.69 and 718.12 +/- 16.48, respectively (Table 3). An increase or decrease in the SASA values is indicative of an impact on the protein structure. Accordingly, the E355K mutation with a higher SASA value resulted in an enlarged solvent-accessible surface area in the E355K structure.Figure 5Rg and SASA plots of wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen following MD simulations (500 ns). (A) Rg values corresponding to the fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes. The x-axis represents time in ns, while the y-axis represents Rg values in nm. (B) SASA values corresponding to the fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes. The x-axis indicates time in ns, while the y-axis indicates area in nm2.Compactness and flexibility of the protein structure are reciprocally connected to each other. Rg and SASA were calculated to study the accessible surface area of mutant structures for the solvent. Kernel density estimation plots of Rg and SASA for wild-type, E355K, and G401C αIIbβ3 structures were plotted, which revealed significant differences in the mutant structures when compared to the wild-type (Fig. 6).Figure 6KDE plots of Rg and SASA are represented together as collective variables for fibrinogen-bound wild-type (A), E355K (B), and G401C (C) αIIbβ3 complexes.Intermolecular H-bondThe hydrogen bonds present in a protein are responsible for maintaining its structure and determining its binding specificity. Intermolecular hydrogen bonds were observed with respect to the wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen which indicated changes in interactions after 350 ns in the mutant complexes when compared to the wild-type (Fig. 7).Figure 7Graphical representations of H-bond interactions of wild-type, E355K, and G401C αIIbβ3 structures bound to fibrinogen following MD simulations (500 ns). The x-axis represents time in ns, while the y-axis represents number of H-bonds.Secondary structure elementsMutations causing changes to the secondary structure of the fibrinogen-bound wild-type and mutant αIIbβ3 complexes were evaluated. The structural behavior of a protein may be influenced by the proportion of secondary structure elements, like α-helixes, β-sheets, turns, etc. Overall, the E355K and G401C complexes had slightly more of these structures, mainly β-sheets, when compared to the wild-type (Table 4).Table 4 Secondary structure elements in fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes.E355K affected fibrinogen bindingBinding free energy was calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach to study the energy association between the αIIbβ3 and fibrinogen structures throughout the MD simulation period. Although the average binding energies for the E355K and G401C structures were found to be lower when compared to the wild-type structure (Table 5A), binding energies associated with active residues involved in ligand binding were higher for these mutant structures when compared to that of the wild-type (Table 5B).Table 5 A. Overall binding energies of fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes. B. Hotspot interaction binding energies of fibrinogen-bound wild-type, E355K, and G401C αIIbβ3 complexes.Table 5 was divided into parts a and b. It is important that tables are numbered in ascending numerical order: 1, 2, and 3. Since such table cannot be separated because of a main caption, it was merged and referred as Table 5. Please check if the modified presentation is appropriate. Otherwise kindly advise us on how to proceed.
