Mutation screening
A total of 177 single nucleotide polymorphisms (SNPs) obtained from the various databases were found within amino acid residues 1-451 of the αIIb subunit corresponding to the b-propeller domain (Table S1).
Pathogenicity analysis
All 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 analysis
Mutations 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 analysis
Amino acid residues that are highly conserved during the course of evolution are unlikely to be mutated. Likewise, conservation analysis was done for all the identified mutations and only those that involved highly conserved residues were studied further. Only nine mutations had occurred in highly conserved regions as indicated (Fig. 2). Although these mutations were reported to be functionally important, structural changes caused by them have not been studied in detail.
Molecular docking studies
Some 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 αIIbβ3 structures with lower binding energy scores were found to form highly stable complexes with greater binding affinity. Among the five mutations, E355K and G401C had the lowest 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.
MD trajectory analysis
E355K compromised stability as compared to G401C
RMSD 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 less 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 less as compared to that of the wild-type (Table 3).
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 indicate 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.
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).
Intermolecular H-bond
The 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 350ns than the mutant complexes when compared to the wild-type (Fig. 7).
Secondary structure elements
Mutations 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 G401S complexes had slightly more of these structures, mainly β-sheets, when compared to the wild-type (Table 4).
E355K affected fibrinogen binding
Binding 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. The average binding energy for the E355K structure was found to be much lower when compared to both the wild-type and G401C structures (Table 5).