Protein residue network analysis reveals fundamental properties of the human coagulation factor VIII

Abstract

Hemophilia A is an X-linked inherited blood coagulation disorder caused by the production and circulation of defective coagulation factor VIII protein. People living with this condition receive either prophylaxis or on-demand treatment, and approximately 30% of patients develop inhibitor antibodies, a serious complication that limits treatment options. Although previous studies performed targeted mutations to identify important residues of FVIII, a detailed understanding of the role of each amino acid and their neighboring residues is still lacking. Here, we addressed this issue by creating a residue interaction network (RIN) where the nodes are the FVIII residues, and two nodes are connected if their corresponding residues are in close proximity in the FVIII protein structure. We studied the characteristics of all residues in this network and found important properties related to disease severity, interaction to other proteins and structural stability. Importantly, we found that the RIN-derived properties were in close agreement with in vitro and clinical reports, corroborating the observation that the patterns derived from this detailed map of the FVIII protein architecture accurately capture the biological properties of FVIII.

Figure 1

The FVIII Residue Interaction Network (RIN). (a) Each residue of the FVIII structure is represented as a node in the RIN. Two nodes are connected if either their main- or side-chains are close to each other (less than ~ 5 Å). Note that the RIN does not keep the three dimensional positional information of the domains or residues. The centrality of each node can be calculated based on the number of residues they interact (degree), whether they serve as bridges for groups of residues that would not be connected otherwise (betweenness), and are located in a position that requires few ‘steps’ to reach every other node in the network (closeness). Image created using the structure 2R7E (Ref.) and Chimera 1.14 (Ref.). (b) Several measures display a moderate to strong Pearson correlation to each other, despite being calculated using different underlying principles.

Figure 2

Centrality measures and mutagenesis results. (a) Alanine mutations on residues of the A2 and C2 domains that are more central in the FVIII RIN caused a reduction of the FVIII co-factor activity, measured by a chromogenic assay measuring thrombin formation^,. (b) A similar effect is observed for the secretion and expression of the mutant constructs; here mutations at the central residues show a significant reduction in the expression/secretion levels, measured using the ELISA assay^,. In all cases, the boxplots depict the median (center line), the first and third quartiles (lower- and upper-bounds), and 1.5 times the inter-quartile range (lower- and upper whiskers). Each dot in the plot is an amino acid mutation (i.e., an in vitro alanine mutant construct). Unpaired, two-sided Wilcoxon test (***Indicate p-values < 0.001; **p-value < 0.01; *p-value < 0.05).

Figure 3

Machine learning framework and predictions. (a) The accuracy of both individual classifiers as well as the ensemble was calculated based on its correct classification of alanine mutations on the A2 and the C2 domains, using a 10 cross-fold validation (“Methods”). The variation in the accuracy values is due to the relatively small input (344 instances). The bars depict mean values and error bars, the standard deviation. (b) The predicted chromogenic activity outputted by the classifiers were only moderately correlated (Pearson’s correlation coefficient, p-value < 0.01). (c) In general, the FVIII mutant constructs with chromogenic activity similar to the wild-type form received high scores from the classifier ensemble. Likewise, low-activity mutants correctly received low scores. The boxplots depict the median (center line), the first and third quartiles (lower- and upper-bounds), and 1.5 times the inter-quartile range (lower- and upper whiskers). Each dot in the plot is an amino acid mutation (i.e., an in vitro alanine mutant construct). Unpaired, two-sided Wilcoxon test (***Indicate p-values < 0.001). (d) Predicted chromogenic activity mapped into the FVIII structure (Supplementary Table S2 lists all predicted values). (e) The relation between the predicted chromogenic activity and the relative surface exposure of the residues of the A1, A3, and C1 domains, indicating that perturbations to the ~ 10%–20% most buried residues (within the 0.1–0.2 range) will likely result in a considerable reduction of the chromogenic activity of the mutant construct. Each dot represents one amino acid from the A1, A3 and C1 domains. Image created using the structure 2R7E (Ref.) and Chimera 1.14 (Ref.).

Figure 4

Critical residues in FVIII. (a) Depicted are the degree and betweenness of all residues of the FVIII RIN, and their assignment to groups that reflect their centrality characteristics. Each dot is one residue from the RIN. (b) The location of the different groups of residues on the FVIII structure. Images created using the structure 2R7E (Ref.) and Chimera 1.14 (Ref.). (c) The relative surface exposure of the key residues identified using the FVIII RIN centrality measures. (d) The closeness of each residue of the FVIII RIN, colored according to the domain where they are located. (e) Boxplot summarizing the closeness centrality of the residues of each FVIII domain. The boxplots depict the median (center line), the first and third quartiles (lower- and upper-bounds), and 1.5 times the inter-quartile range (lower- and upper whiskers).

Figure 5

FVIII binding sites and neighboring residues. (a) Location of known FVIII binding sites (pink) and their immediate neighboring residues (blue) in the FVIII structure and in the RIN. Image created using the structure 2R7E (Ref.) and Chimera 1.14 (Ref.). (b) Comparison of the centrality measures of the residues reported to be part of a binding site and their immediate neighboring amino acids. The boxplots depict the median (center line), the first and third quartiles (lower- and upper-bounds), and 1.5 times the inter-quartile range (lower- and upper whiskers). Each dot in the plot is a node from the FVIII RIN (i.e., an amino acid). Unpaired, two-sided Wilcoxon test (***Indicate p-values < 0.001).