In silico analysis of Glanzmann variants of Calf-1 domain of α_IIbβ₃ integrin revealed dynamic allosteric effect

Abstract

Integrin α_IIbβ₃ mediates platelet aggregation and thrombus formation. In a rare hereditary bleeding disorder, Glanzmann thrombasthenia (GT), α_IIbβ₃ expression / function are impaired. The impact of deleterious missense mutations on the complex structure remains unclear. Long independent molecular dynamics (MD) simulations were performed for 7 GT variants and reference structure of the Calf-1 domain of α_IIb. Simulations were analysed using a structural alphabet to describe local protein conformations. Common and flexible regions as well as deformable zones were observed in all the structures. The most flexible region of Calf-1 (with highest B-factor) is rather a rigid region encompassed into two deformable zones. Each mutated structure barely showed any modifications at the mutation sites while distant conformational changes were observed. These unexpected results question the relationship between molecular dynamics and allostery; and the role of these long-range effects in the impaired α_IIbβ₃ expression. This method is aimed at studying all α_IIbβ₃ sub-domains and impact of missense mutations at local and global structural level.

Introduction

In humans, the Integrins protein superfamily consists of 24 heterodimeric receptors resulting from different combination of 18 α and 8 β subunits. Integrins are highly dynamic glycoprotein (GP) involved in cell-cell or cell-matrix interactions¹. Upon activation the integrin α_IIbβ₃ binds plasmatic fibrinogen leading to platelet aggregation and thrombus formation (Primary haemostasis). Some well-known human integrin structures are: α_xβ₂, α_vβ₃ (cell-matrix adhesion) and α_IIbβ₃ (cell-cell adhesion). α_v can associate to β_1,3,5,6,8 subunits while α_IIb, that is specific to platelets and megakaryocytes cells, only associates to β₃ ¹. The α_IIbβ₃ structure is organized into 3 distinct regions; an N-terminus extracellular ectodomain, a single spanning transmembrane (TM) region and a C-terminus cytoplasmic region. The cytoplasmic region in α subunit is very swift (~20 residues) while in β subunit it extended up to 46 residues in length and constitutes an important node for signalling. Both α and β TM regions are single spanning, and consist of 22 residues each. The ectodomain is relatively huge with 959 and 693 residues in α and β subunits respectively. Figure 1 depicts transition steps from the inactive conformations of α_IIbβ₃ (crystallized closed structure) to its theoretical open liganded active form. A complete structure of the open forms of the ectodomain with or without ligand remains to be crystallized. The ectodomain is further divided into four regions: headpiece, knee, legs and tails. The headpiece that carries the ligand-binding site consists of the β-propeller domain of α_IIb and the β-I domain of β₃. The α_IIb β-propeller domain consists of a 7 bladed fold with four Ca²⁺ ions coordinated with β-hairpin loops connecting the antiparallel β-strands (see Fig. 1A). The β-I domain of β₃ mainly consists of α helices and loops with coordinated metal ions Ca²⁺ and Mg²⁺ constituting a MIDAS (Metal Ion Dependent Adhesion Sites) with an ADMIDAS (Adjacent to MIDAS) and SyMBS (Synergistic Metal Binding Site). These sites play critical role in opening the α_IIbβ₃ binding site and helps in ligand binding². Following β-propeller, the α_IIb leg is composed of the Thigh domain, the Genu (knee), the rigid Calf-1 and Calf-2 domains. The short loop of α_IIb Genu coordinates with a divalent calcium³. This metal ion might help in stabilizing Calf-1 and Thigh domains interface during the opening of the structure following the activation process (angular shift at Genu). The α_IIb leg is rigid and provides a framework to the entire ectodomain. The β₃ β-I domain of the headpiece is followed by the Hybrid, PSI, and 4 IEGFs (Integrin Epidermal Growth Factor) domains. A short knee joined the IEGF-1 with IEGF-2 domains. The β₃ leg consists of IEGF-2 to IEGF-4 whose C-terminus ends in the ankle domain (tail). α_IIb and β₃ transmembrane and cytoplasmic domains are not shown. The activation state of α_IIbβ₃ is tightly controlled by inside - out signalling resulting from platelet activation by multiple exogenous factors (physiological plasmatic agonists, exposed sub-endothelial matrix …). The α_IIb headpiece opens up creating an angular shift between Thigh and Calf-1 domains (Fig. 1C) meanwhile, the β₃ leg and tail remains parallel to the α_IIb leg. Thereafter, opening of the β₃ headpiece pulls the β₃ legs outward resulting into an extended open conformation (Fig. 1D,E) that can bind plasmatic fibrinogen at the MIDAS, which is constituted by elements of both the headpieces^{4, 5}. Finally, fibrinogen binding leads to outside-in signalling.

Figure 1

Closed to open transition of α_IIbβ₃. (A) The closed form of α_IIbβ₃ ectodomain, with Calf-1 domain highlighted in green. Rest of the structure is depicted in dull grey to bring clarity. Structural organization of ectodomain is labelled. The rainbow schema of colours on secondary structures represents the α_IIbβ₃ structure. Regions in green-blue spectra mark the α_IIb subunit and yellow-red spectra mark the β₃ subunit. (B) Closed inactive form of α_IIbβ₃. The structure is bent along the plane of knee domains. (C) Extended α_IIb headpiece with β₃ leg resting alongside the α_IIb leg. (D) Extended β₃ conformation: The β₃ headpiece has intrinsic conformational changes at C-terminus leading to an outward pull of the β₃ leg. (E) Extended α_IIbβ₃ conformation: In the last stage, β₃ headpiece pulls out creating a ligand-binding cavity between the two headpieces. Mg⁺⁺ constituting MIDAS can be seen as a green sphere in the cavity, while the ligand Fibronectin (dull grey dots) approaches the glycoprotein. All metal ions are shown as solid spheres with golden representing Ca²⁺ while green representing Mg²⁺. Polysaccharides (N-acetyl glucosamines and Mannose) are shown in ball and stick representations. Please notice that the open forms had been modelled from the closed structure according to expected conformations using Modeller_v_9.16 and images are generated by PyMol_v_1.7.0.

The human integrin α_IIbβ₃, a platelet surface fibrinogen receptor, is responsible for platelets aggregation, a key process in primary haemostasis and thrombus formation⁶. Defective primary haemostasis leads to two severe life-threatening bleeding disorders: (1) Glanzmann thrombasthenia (GT), a rare autosomal recessive genetic disease associated with impaired α_IIbβ₃ expression and/or function⁷; and (2) Fetal/neonatal alloimmune thrombocytopenia (FNAIT) results from fetal/neonatal platelet destruction by maternal antibodies in mothers lacking an alloantigen inherited from the father. FNAIT clinical consequences range from no symptoms to intracranial haemorrhages with a risk of neurological sequel and/or fetal/neonatal death⁸.

Both diseases result from α_IIb and β₃ gene polymorphisms. In GT, more than 300 mutations have been identified in α_IIb or β₃ genes. Most of them are reported in https://sinaicentral.mssm.edu/intranet/research/glanzmann. In FNAIT, neither the expression nor function of α_IIbβ₃ is affected but missense mutations results in amino acid (aa) substitutions that define Human Platelet Antigens (HPA). All human platelet alloantigens are described in the HPA database (http://www.ebi.ac.uk/ipd/hpa/). The effects of these amino acid substitutions on α_IIbβ₃ structure remain largely unknown. We are interested in understanding how amino acid substitutions in GT can impact the Calf-1 domain of α_IIbβ₃ structure and its structural dynamics.

Very few structures of α_IIbβ₃ have been crystallized and only one contains the whole α_IIbβ₃ ectodomain (PDB id 3FCS⁹) in closed conformation (Fig. 1B). Using this structure, we showed that the β3 Lys253Met GT mutation impaired key ionic interactions between the α_IIb β-propeller and the β₃ β-I like domain¹⁰. However static models cannot depict all mutation-induced effects on a highly dynamic structure.

In our previous studies, molecular dynamics (MD) simulations were used to compare the structures of the β3 L33 and P33 forms¹¹. The L33P substitution located in the PSI domain is responsible for the HPA-1 system, clinically the most important one in Caucasian populations. A third form with a Valine in position 33 of β3 was also studied¹². Although the 3 variants mostly shared common conformations, the P33-β3 variant presented a higher mobility and specific conformations of IEGF-1, IEGF-2, and PSI domains. The L33V substitution mainly displaced a dynamic equilibrium between common structures that could explain a variable reactivity of different anti-HPA-1a sera with the two β3 forms.

Using new strategies in MD, we studied the effect of 7 variants of the α_IIb Calf-1 domain known to impair α_IIbβ₃ integrin expression in GT. Unexpected long-range structural effects of mutations were discovered. Furthermore, our results raised the question about a possible role of these allosteric effects in the impaired α_IIbβ₃ expression.

Methods

Structural data

The α_IIb Calf-1 domain was extracted from a 2.55 Å resolution crystal structure of the α_IIbβ₃ integrin (PDB code 3FCS⁹). Calf-1 is a domain of 141 residues [positions 603–743] (see supplementary videos 1 and 2). It is a mainly-beta sandwich protein with an immunoglobulin-like topology as described in CATH database (CATH number: 2.60.40.1510, http://www.cathdb.info/version/latest/domain/3fcsA03)¹³. Some missing atoms in side chains of residues 667 and 668 were completed using Modeller software v.9.14^{14, 15}. The seven GT aa substitutions were introduced in Calf-1 structure by in-silico mutagenesis using PyMOL software¹⁶ and the SCWRL method¹⁷. The effects of all mutations were studied exclusively.

Molecular Dynamics

MD simulations were done using GROMACS 4.5.7 software¹⁸ with the OPLS-AA force-field¹⁹. WT and variant forms of Calf-1 were soaked in a rhombic dodecahedral simulation box with TIP3P water molecules and neutralized with Cl^- ions. The MD protocol had been used in our previous works^{11, 12}. After 1 nsec of equilibration (with position restraints on the protein), each system was simulated through 11 independent dynamics for a total of 850 nanoseconds (5 × 50 nsec + 6 × 100 nsec). Molecular conformations were saved every 100 psec for downstream analysis. The first 5 nsec of each MD simulation were discarded as the residues at the extremities. Trajectory analyses were done with the GROMACS software, in-house Python and R scripts. Root mean square deviations (RMSD) and root mean square fluctuations (RMSF) were calculated on Cα atoms only. Residues interactions were analysed using the online tool PIC (Protein Interactions Calculator)²⁰.

Protein Blocks analysis

Protein Blocks (PBs) are a structural alphabet composed of 16 local prototypes²¹. Each specific PB is characterized by the φ, ψ dihedral angles of five consecutive residues with each PB assignment focused on the central residue. Obtained through an unsupervised training approach and performed on a representative non-redundant databank, PBs give a reasonable approximation of all local protein 3D structures²². PBs are very efficient in tasks such as protein superimpositions²³ and MD analyses²⁴. They are labelled from a to p: PBs m and d can be roughly described as prototypes for α-helix and central β-strand, respectively. PBs a to c primarily represent β-strand N-caps and PBs e and f representing β-strand C-caps; PBs a to j are specific to coils; PBs k and l to α-helix N-caps while PBs n to p to α-helix C-caps. PB²⁵ assignment was carried out using our PBxplore tool at GitHub²⁶.

PB assignments are done for each residue of the Calf-1 domain and over every snapshot extracted from MD simulations. The equivalent number of PBs (N _eq ) is a statistical measurement similar to entropy that represents the average number of PBs for a residue at a given position. N _eq is calculated as follows²²:

$$Neq=\exp (-\sum _{x=1}^{16}{f}_{x}\,\mathrm{ln}\,{f}_{x})$$

(1)

where, f _x is the probability of PB x. A N _eq value of 1 indicates that only one type of PB is observed, while a value of 16 is equivalent to a random distribution. To underline the main differences between the wild-type (WT) and a variant for each position, ΔN _eq value is computed. ΔN _eq is the absolute difference between corresponding N _eqs .

However, a same ΔN _eq value can be obtained with different types of blocks in similar proportions. To detect a change in PBs profile, a ΔPB value was calculated. It corresponds to the absolute sum of the differences for each PB between the probabilities of a PB x to be present in the WT and the variant forms (x goes from PB a to PB p). ΔPB is calculated as follows:

$${\rm{\Delta }}PB=\sum _{x=1}^{16}|({f}_{x}^{WT}-{f}_{x}^{\mathrm{var}})|$$

(2)

where, f ^WT _x and f ^var _x are the percentages of occurrence of a PB x in respectively the WT and the variant forms of Calf-1 structures. A value of 0 indicates perfect PBs identity between WT and variant, while a score of 2 indicates a total difference.

Calf-1 variants

The seven Calf-1 domain variants studied herein were involved in GT and are reported in the GT database (https://sinaicentral.mssm.edu/intranet/research/glanzmann). Variants L653R²⁷, L721R (only reported in the GT database), L721V²⁸, R724P^{28, 29}, R724Q³⁰, and P741R²⁹ severely impaired α_IIbβ₃ expression (less than 5%) while variant C674R^{28, 30,31,32} allowed a 10% residual expression (type I and II GT³³).

Results

Structural analysis of the Calf-1 domain

Calf-1 domain extends from residues 603 to 743 of the α_IIb integrin subunit. This domain is an all beta structure adapting an Immunoglobulin-like Beta-sandwich fold¹³ with 9 consecutive β-strands connected by 8 loops (Fig. 2, the loops position is provided in Fig. 3D). Loops 1 and 10, located at the N- and C-terminals of Calf-1 connects it with N-ter Thigh and C-ter Calf-2 domains, respectively. RMSD from all MD simulations reach a steady state at 50 nsec (Figure S1) that is maintained in longer runs of 100 nsec indicating stable and reproducible independent dynamics.

Figure 2

Ribbon model of the Calf-1 domain showing the location of the studied variant residues. β-strands are coloured in green and loops in yellow. Variant residues are identified as red balls. N-ter and C-ter ends are shown as yellow balls.

Figure 3

Comparison of the protein flexibility of Calf-1 through different metrics. 3D structures of Calf-1 domain represented through (A) B-factor values, (B) RMSF values, and (C) N _eq values. Local structure is ranked from rigid (thin blue line, a value of 0.0) to flexible (thick red line, a value of 4.0). Residues with completed missing atoms are in grey in the B-factor cartoon (A). (D) The Calf-1 amino acid sequence is placed in regard to its secondary structures assignment and to protein flexibility according to the B-factor, the RMSF or the N _eq values. Blue, green, yellow, orange and red colours scale the structure from rigid to flexible. The loops are: loop 1 (size: 9, positions 603–611), loop 2 (size: 10, positions 620–629), loop 3 (size: 7, positions 640–646), loop 4 (size: 4, positions 653–656), loop 5 (size: 8, positions 665–672), loop 6 (size: 6, positions 678–683), loop 7 (size: 6, positions 690–695), loop 8 (size: 8, positions 708–715), loop 9 (size: 11, positions 725–735), and loop 10 that begins at position 742.

According to the high B-factor values obtained from crystallographic data, loops 2, 3, 4 and 5 are the most flexible regions of Calf-1 (Fig. 3A). Residues 622, 643, 710 and residues 667/668 (of loop 5 that contains missing atoms) presented the highest B-factor values in their respective loops. On average β-strands are more rigid than loops^{34, 35}, although some of their residues represent relatively high B-factor values. In our study, the B-factors seemingly escape the influence of crystal packing contacts. However, it is known that B-factors are strongly influenced by the crystal packing of the structure³⁶. Some protein moieties very flexible in solution might seem to be rigid only because they are involved in the solid-state packing.

RMSF values computed from MD simulations measure the mobility of each residue around its median position in the structure and allow assessing protein flexibility (Fig. 3B). High RMSF values are often associated with loops and sometimes with C-ter of β-strands. As defined by high RMSF values, loop 2 (residues 619–620 and 625–626), loop 5 (residues 665–671) and loop 8 (residues 711–713) are flexible regions, with loop 5 being the most flexible. The rest of the structure is more rigid. RMSF and B-factors values are correlated for loops 2, 5 and 8 (Fig. 3D). Some points are noteworthy: (a) the limits of flexible positions can show some little differences between RMSF and B-factor and (b) loop 3 is associated to high B-factor but low RMSF values although it binds a Ca²⁺ (not included in MD simulations) in the crystal structure. Similar correlation between B-factor and RMSF values had been previously reported³⁷.

Figure 3B–D indicate a good correlation between RMSF and N _eq values. Indeed, highest N _eq values are associated to flexible regions (as defined by B-factor and RMSF) with residues K678-T682 (loop 6) and N709-E712 (loop 8), but also with T619 (loop 2). Expectedly, some regions can show higher N _eq for some residues; G641-G643 (loop 3) and S728-N730 (loop 9). On the other hand, highly flexible region can also represent high local rigidity in terms of PBs, for instance, residues V666-F669 and E670 in loop 5 (Figs 3C and 4). Direct comparison of RMSF and N _eq values (Fig. 4A) clearly shows that E667 represents a high RMSF but a low N _eq . This can be explained by its PB distribution (Fig. 4B): E667, G668 and F669 representing the highest RMSF values (and also B-factors), mainly adopted the PB sequence “hia” with respective occurrences of 86.2, 82.9 and 61.6%. A series of PB “hia” is a classical loop conformation but this region (in blue rectangle on Fig. 3C) maintains a single conformation and is not really flexible. This apparent discrepancy can be explained by the insertion of the rigid stretch E667-F669 in a larger flexible (or more precisely deformable) loop N665-L672. Interestingly our results revealed that a locally rigid aa stretch (few possible conformations/low N _eq ) can be a part of a large mobile loop involved in the global structural motions of the protein (high RMSF).

Figure 4

Local rigid conformation in a deformable loop, low N _eq versus high RMSF. (A) Superimposed RMSF and N _eq values (red and blue curves respectively) from residues N665 to G668, (B) The WebLogo⁴⁹ indicates the frequency of occurrences with respect to the PBs adopted (size of the letter) by a residue in MD simulations. Here, residues V666 to F669 mainly adopted the PBs profile “ehia” corresponding to low N _eq for them. (C) 3D model of the Calf-1 domain and the frame magnified of two adopted by the loop conformations (red and yellow worm-lines) carrying the residue E667 (in blue) that keep a rigid structure relative to the mobile loop.

Overall, our results showed a good correlation between experimental data (B-factor), RMSF and N _eq obtained from MD simulations. Although some discrepancies did exist, they are explained by local structure singularities. As expected in an all-Beta domain, rigid β-strands are linked by flexible loops.

The selection of OPLS-AA force field was made in regards to our previous works on integrins^{11, 12}. To assess the effect of the force field, simulations were also performed on WT with Gromos 46a7 and Gromos 54a7. For the latter, 11 independent simulations were performed with the protocol used with OPLS-AA. No significant global or local differences were observed.

Structure comparisons between GT variants and WT Calf-1

The α_IIbβ₃ integrin was cut into compact structural domains through Protein Peeling³⁸ that correlate the delineations found in literature⁹. As shown in Fig. 2, the variant residues studied are mostly located at β-strands presenting low flexibility with the exception of residue 653 localized near the β-strand 3 C-ter.

As for the WT system, the 7 variants were studied with 11 independent MD simulations performed to a complete timing of 850 nsec and with parameters similar to Jallu et al.¹². Each system reached a plateau after 5 nsec with an average RMSD of 2 Å (beginning of loop 1 and end of loop 10 excluded). All energetic and geometric parameters showed a good evaluation for the 77 different simulations used in this study, e.g. no clashes found. The Calf-1 domain stayed consistent during the whole dynamics.

Average RMSF from each variant and the WT were comparable (Fig. 5). The most important variations observed in loop 2 (V625), loop 5 (E670), loop 8 (A713) and loop 9 (N732) did not lead to disordered patterns. Some variants showed specific higher or lower RMSF for some restricted positions like for C674R and L721V variants (Fig. 5). As seen with the WT system, RMSF can be confused with deformability. To resolve this aspect, we have used PBs analyses of the MD trajectories.

Figure 5

Calf-1 RMSF of the different systems. By comparison, Calf-1 variant structures mainly behaved like the WT form (black curve). The noisy peaks for the N-ter first residues were discarded in the majority of the analyses since, in nature they lay at conjunction to the neighbouring domain.

PBs analyses revealed striking local structure alterations, but distant from the variant sites

For clarity, results are detailed for only 3 variants R724Q, L653R and C674R that are representative of all behaviours observed for the 7 variants.

R724Q

This aa variation is located at β-strand number 8. In regards to the WT structure (Figs 6A and S2A), the highest N _eq differences are at S621 (beginning of loop 2), A644 (loop 3) and L710 (loop 8). These loops that are naturally flexible are even more so in the variant. Conversely, residues L624 to D628 have a lower N _eq value thus indicating that loop 2 represents a dual behaviour, with increased deformability at its beginning and enhanced stability in its C-ter part. Surprisingly, the mutant residue Q724 (β-strand 8) conserved the same N _eq (Fig. 6A) with a low ΔPB of 0.09 (Fig. 6B) indicating that local β-strand conformation is conserved, i.e. PB d.

Figure 6

Variant R724Q. (A) N _eq values of residues from the WT system (black curve) and of the R724Q variant (red curve). Positions of the residues 724 and S621 that presented the highest RMSF alteration are respectively indicated by blue and green dots lines. (B) Curve of the ΔPB values computed from the difference between the two systems. (C) PB maps from residues 722 to 726 for the WT (left) and the variant (right) systems. (D) PB maps for residues 619 to 623. Color scales indicate the frequency of occurrences of the PBs in the map. (E) Molecular interactions made by the residues 724 in the WT and the variant systems (left and right cartoons respectively). (F) Molecular interactions made by the residues 621 in the WT and the variant systems (left and right cartoons respectively). Residues 724 and S621 are shown as cyan balls in the WT form, and as light cyan balls the variant. Orange balls indicate residues that conserved their interactions with the residues 724 or S621 while magenta balls correspond to residues with modified interactions. A cartoon of the Calf-1 domain shows the respective locations of the residues 724 and 621.

Regarding the structure, the polar amino acid arginine contains a longer aliphatic side-chain than glutamine, an uncharged hydrophilic polar amino acid. Q724 conserves the backbone - backbone interaction with E648 as observed with R724 (β-strand 3, see Fig. 6E). Beside, Q724 lost the ionic bond and the side chain - side chain interactions with E648 but made new hydrogen bonds through side chains interactions with E722. This showcases a classic example of structural compensation that maintained the local conformation of the residue through different interactions.

The highest ΔN _eq (2.71) that is also associated with the highest ΔPB (0.57), is observed for S621 (Fig. 6A,B). S621 is located at the opposite side of the domain in reference to residue 724 (Fig. 6E). In the variant structure, S621 mostly remained in a PB d (i.e., β-strand) conformation with however, a decreased frequency of occurrences. Besides, downstream P622 and L623 presented some lost conformers with increased frequencies of PBs e and h respectively. Very few typical backbone - backbone interactions of S621 with L623 and backbone - side chain interactions with N629 are replaced by a single bond between side chains with N629. Adding to this high mobility, S621 did not do consistent and sustainable interactions. This behaviour is amplified in the Q724 variant and the most stable residue S621 in a naturally flexible region (loop 2), became one of the most deformable position.

L653R

This GT variant results from a L653R substitution in loop 4. The highest N _eq variations (Figs 7A and S2B) affected residues G620-P622 (loop 2), V630-L631 (β-strand 2), E646 (loop 3), R671 (loop 5) and L710 (loop 8). As observed with the R724Q substitution, residues G620-L623 gained slightly more flexibility. Conversely, residues L624-D628 showed increased flexibility but with a limited impact (ΔPB 0.23 on average) on the most frequent PBs (PB e for L624, h for V625 and i for G626 in Fig. 7B).

Figure 7

The variant L653R. Panels (A and B) respectively show the N _eq and the ΔPB curves of the L653 (WT) and R653 (variant) systems. Panels (C,D) respectively show the PB maps of residues 651 to 655 and 677 to 681 with the WT at the left and the variant form at the right. (E) Molecular interactions made by the residues L653 or R653 and (F) by E679. For colour scales and residue presentation, see the legend of Fig. 6.

The mutated residue in position 653 (loop 4) was not subjected to any N _eq modification. It conserved a strong local structural stability (Fig. 7C) similar to its direct environment. The PB series at this position “dddeh” is even slightly more common in the variant than in the WT (64% and 59%, respectively). In the R653 variant, the 8 hydrophobic bonds of L653 disappeared in favour of new interactions between the R653 backbone and A657 and E676 side-chains (Fig. 7E). The backbone – backbone interaction with R683 was conserved. The mutation zone showed no conformational change as the loss of important specific interactions were partly compensated by new ones. Over 9 original interactions only 1 is conserved while 3 are created.

Q679 (loop 6) is a very interesting case where ΔN _eq was negligible while the ΔPB was the highest (0.78). The most frequent PB b (N-cap of β-strand) was replaced by a PB h (loop structure) in regards to their frequency of occurrences (Fig. 7D). Hydrogen interactions with T682 and K677 are retained but the backbone - backbone interaction with E681 was lost and replaced by side chain and ionic side chain interactions with R724 in loop 9. In the variant structure, this region has high fluctuations in PBs, mainly associated to loops that even affected the C-ter of the β-strand 5 located the above loop 9.

C674R

This variant is associated with a C674R substitution in β-strand 5. We observed a variation of N _eq profile (Figs 8A and S2C) similar to that occurring in the R724Q substitution (see previous section). Loop 2 presented the same increased deformation at its beginning (S621), followed by a stiffening in its centre (residues L624-D628). The same PB series “ehiac” (L624-D628) is found in greater proportion than in the Q724 and R674 variants, reinforcing the local stiffening of the loop in this region.

Figure 8

The variant C674R. Panels (A and B) respectively show the N _eq and the ΔPB curves of the C674 (WT) and R674 (variant) systems. Panel C show the PB maps of residues 669 to 676 for the WT (above) and the variant forms (below). (D) Molecular interactions made by the residues C674 or R674 and (E) R671. For colour scales and residue presentation, see the legend of Fig. 6.

The main destabilization was far upstream of residue 674 (Fig. 8C). With the C674R substitution, the residue 674 not just lost its covalent disulphide bond with C687 located at the end of β-strand 6, but also its aromatic interaction with Y659 in β-strand 4 (Fig. 8D). However, the mutated R674 made an ionic bond with E688 located at end of β-strand 8 that strengthened a backbone – backbone interaction. The 80% frequency of PB d (the highest) in WT decreased to 49% in the variant. Surprisingly, N675 and Q676 located downstream the substitution remained structurally stable with similar PB occurrences.

The highest N _eq variation affected R671 as shown by the strongest ΔN _eq (5.02) and ΔPB (0.91). The side chain of R671 is mainly exposed at the domain surface and forms a single ionic interaction with the neighbouring E670, like in WT. But in the variant conformation, the R671 side-chain can occasionally turned toward loop 8 to make ionic side chain interactions with E688 (Fig. 8E). The frequency of PB d (the highest) drastically decreased in the variant leading to an increased disorganization of the neighbourhood.

Experimentally, the C674R mutation severely impaired the α_IIbβ₃ complex expression with only 10% expressed at the surface of the patient’s platelets and transiently transfected CHO cells³¹. The C674R mutation did not impair pro-α_IIb synthesis but affect the stability of the complex that is not correctly matured and/or expressed at the cell membrane.

Other variants

These MD simulations on Calf-1 domain allowed demonstrating more or less pronounced structural changes depending on the variants under study. The C674R and P741R variants presented conformational changes at the mutated site. In the case of the C674R substitution, the resulting loss of the disulphide linkage relaxes the structure and introduces significant structural alterations (Figs 7 and 8). The proline is the aa known to cause the most drastic change in conformations³⁹. Indeed, the P741R substitution (Fig. 9) inverse the PB profile going from 55% of PB d (β-strand) and 29% of PB f (C-cap of β-strand) to 24% of PB d and 59% of PB f (Figure S3). This case was associated with a low ΔN _eq (0.15, Figure S4A) while the ΔPB was high (0.70). In P741R substitution two hydrophobic interactions were lost and R741 formed ionic and side chain - side chain interactions shortening the β-strand. Note that residue 741 is located at two residues from the C-term of Calf-1 and is normally in contact with the Calf-2 domain. Thus, the absence of the neighbouring domain in our MD simulations can impact our observations. To solve this problem, MD simulations of combined domains are currently under study.

Figure 9

Comparative study of the ΔPB values for all WT-variant pairs. Histogram schema presenting the ΔPB value computed for each residue position (abscissa) from each variant and the WT systems. Green triangle indicates the aa variation position while the purple one position shows the position of maximal ΔPB. Residues from loops 2, 3 and 8 presenting common high ΔPB for all variants are boxed.

In the remaining 5 variants studied, compensation mechanisms were also observed. Most interactions formed by WT residues are replaced by new ones, allowing conservation of the local structure. Surprisingly, regions displaying significant changes (high ΔPB) are distant from substitution sites without any contact/interaction with the substituted aa. These regions contribute towards increasing the deformability and are usually located at interfaces adjacent to neighbourhood β-propeller, Calf-2 or Thigh domains. These results depict changes resulting from substitutions in distant regions suggesting long-range mechanism to be at play.

Different variants with common mutation sites

L721R and L721V showed quite different results (Fig. 9). Compared to L721R, the L721V substitution had very little impact on RMSF, apart for the end of loop 8, a highly flexible region. This is particularly true for E712 (loop 8), whose ΔN _eq were respectively of 3.33 and 0.39 in R721 and V721 variant forms (Figures S4B and S5). Residue 721 and E712 are remote (separated by β-strand 8 and a part of loop 9), and E712 can interact with the β-propeller domain. Loop 8 and especially E712 are already naturally mobile. Locally, V721 also presented a lower impact than R721 that has a higher overall ΔN _eq . These observations sound logical as Leucine and Valine share similar hydrophobic aliphatic side-chains (a single methyl group of difference), mitigating the impact of the L721V substitution with respect to arginine, which is positively charged.

R724P and R724Q

The previously described R724Q substitution (see above) presented a very pronounced impact compared to others and induced the dual deformability/rigidity alterations of loop 2. This particular phenomenon is also observed in the variant R724P although contributing more stiffness to the site, owing to the backbone structure of proline (Fig. 9). In both cases, the mutated residue did not affect the local conformation (ΔN _eq and ΔPB are identical). The P724 variant mainly differed from the R724 WT in residues G690 (C-cap of β-strand 6) and N691 (start of loop 7, in contact with the Thigh domain). G690 and N691 have maximal ΔPBs of 0.79 and 0.75, and ΔN _eq of 2.17 and 0.85 respectively. For G690, these values are consistent with a drastic PB j (loop) decreased from 70% in the WT to 23% in the variant, while the occurrences increased for very rare PBs i and p (loop and N-cap of α-helix) (Figure S6). For N691, PB c occurrences decreased from 80% to 45%, while PB a reached 44%; both PBs types being associated with N-cap β-strand.

Considering the 7 variants, some Calf-1 regions are more affected than others (see Figure S6). For instance, loop 5 had certain rigidity in terms of PBs but this area is subject to strong N _eq increment under influence of substitutions involved in Glanzmann thrombasthenia (e.g., case of variant C674R). Loop 8 deformability is also observed to be increased, especially in the L721V substitution. The region from the middle of loop 2 to the middle of β-strand 2 (L624-E632) is also affected, from deformability (L653R, L721R) to rigidity (C674R, R724Q).

Loop 2 (especially residues G620-P622) is particularly a remarkable region with a local variability common to all variants (Figs 8 and S6). The end of β-strand 1 has high ΔN _eq values ranging from 2.06 to 2.71, while ΔPB ranges from 0.54 to 0.71. A direct comparison reveals that PBs a, b and d frequency of occurrences in the WT decreased from 66% to 37% in the variant.

Discussion

This study aimed at investigating the α_IIbβ₃ integrin structure in a context of GT disorder. Previously, two MD studies of GT mutations have been performed, both with very short simulation times. The first study addresses the mutation in a calcium binding site with a single 20 ns simulation for reference and variant sequences⁴⁰. A second study carried on the β3 S189 variant was done with a single simulation of 60 ns⁴¹. Hence, both these studies were very short for sampling the conformational space.

Our team showed the impact of three distinct mutations by a static approach¹⁰. More recently, we have focused on the HPA-1 system characterization with MD simulations 10.8 times longer than the longest study cited herein^{11, 12}. In the present work simulation times were 14.1 times longer and 11 independent simulations were done for each variant to correctly sample the conformational space. To illustrate this, Sammon maps⁴² of the 88 independent MD were done for the 8 systems (WT and 7 variants, Fig. 10). In brief, a Sammon map (SM) is a projection method, which differs from Principle Component Analysis (PCA). SM is an iterative approach that tends to conserve the distances between data within a high-dimensional space into a smaller dimensional space, often a 2D space. A major interest of SM is that it does not overweight the outliers; therefore the projection is really pertinent. SM of MD encoded in terms of PBs allowed comparing distances between MD obtained in a single or multiple systems. Considering first a single system, the limited numbers of simulations in a close vicinity to its barycentre underlined the differences between simulations as observed for the WT (Fig. 10A). Similar dispersion is obtained for each system (Fig. 10B). Two systems with variants at the same position like L721R (green dots)/L721V (yellow dots), and R724P (brown dots)/R724Q (cyan dots) can also lead to somewhat superimposed positions (Fig. 10B). Nonetheless comparing the whole set of positions (or their barycentre) clearly indicate differences in behaviour of MD of each system. It confirmed the necessity to proceed with several independent simulations otherwise the conclusions might be biased.

Figure 10

Sammon map of the MD. (a) The WT reference system and its barycentre, (b) all the variant simulations and the barycentre of each system, and (c) only the barycentres. The MD are shown as dots, and corresponding barycentres as plain circles.

Sammon maps (Fig. 10C) showed that it is also possible with PB encoding to find some relationship between different systems. C674R and P741R were the most distant systems compared with the WT, while the other variants studied are closer to the reference. Interestingly the two variants of residue 721 are not close (yellow /green dots) while the variants of residue 724 (brown/blue dots) are. These examples showed that 2 variations at a same position could lead to similar or different behaviours.

Calf-1 domain is an all-β structural domain⁹. Analysis of both B-factors obtained from crystallographic data and RMSF measured from simulations showed that the more flexible regions are connector loops, as expected. The usage of three different metrics, B-factor, RMSF and PBs related values (N _eq and ΔPBs), provided a more complex yet comprehensive way to analyse protein flexibility locally. Comparison of these metrics revealed some interesting cases and some of these results are worth to be underlined. Loop 5 had the highest B-factor (where some atoms are missing⁴³) and RMSF values. However, low N _eq values indicate that a highly rigid zone forms the core of the loop. This underlines interesting contexts of mobility: rigid stretch encompassed in larger deformable region leads to high mobility (high B-factor and RMSF values) while locally the loop structure did not change (low N _eq values). In the case of loop 2, its central residue P622 is associated to a high B-factor value, but neither high RMSF nor high N _eq are. It is its neighbourhood regions that would have the highest mobility. Other interesting cases are positions V625-G626 (loop 2) and E642-G643 (loop 3) that had antinomic behaviours. Both had low B-factors, even if E642 is interacting in the crystal structure with Ca²⁺ ion. While residues V625-G626 had high RMSF and low N _eq , mimicking the loop 5 behaviour in a lesser extent, residues 642 to 643 had medium RMSF but high N _eq . This last case is more complex: structural variations though associated to several distinct conformations (high N _eq ) were highly local, limiting the RMSF values.

These results underline the interest of N _eq computation in regards to classical measures and reveal the high complexity of MD thus enabling to highlight a single or only few critical residues.

The most surprising result concerns the minimal or absent structural effect observed at the aa variation site. Apart from the two very specific cases of C674R (leading to disulfide bridge disruption) or R724P (replacement by a proline) mutants, in all other studied GT variants, local conformations are maintained through new molecular interactions. Nonetheless such compensatory mechanisms cannot account for impaired αIIβ3 expression associated with the 7 GT variants tested. Another surprising result is that most important alterations are located at long distances from the mutation sites. These alterations increase mobility or deformability of the same adaptable regions (e.g. loops 2, 3 and 8) in all variants, a new concept to biomolecular structural biology as defined by R. Nussinov^{44, 45}. Furthermore although RMSF profiles of the WT and the 7 variants were very similar (Fig. 5), it is particularly surprising that N _eq values of most loops are affected, sometimes drastically (Fig. 9). Only loops 4 and 7 presenting very low N _eq values in the WT form, were least affected.

The 7 variants tested under the present work severely impacted the α_IIbβ₃ integrin expression. Using NN-Splice⁴⁶ and GENSCAN⁴⁷ on-line softwares, the genomic mutations involved in the aa variations studied were not predicted to induce splicing defects (Results not shown). Thence these aa variations should alter the biosynthesis of the α_IIbβ₃ complex by structural alterations. Hence, these variant structures represent common and specific structural effects regarding the WT Calf-1 domain (ΔN _eq and ΔPBs). Observed modifications rather concerns structure dynamics than large structural alterations (loss of interaction¹⁰) as previously seen in other studies. These results raise the possibility that current explanations of GT type I and II phenotypes regarding structure alterations might be reconsidered. In fact our results point out that structural mechanisms leading to an impaired expression of α_IIbβ₃ integrin can be far more complex than expected. It must be noted that our study did not allow identifying which of the observed effects are responsible for the pathogenicity of the aa variations tested. Interestingly, two mutations at the same site (L721R/L721V and R724P/R724Q) can lead to similar or different effects, underlining that long-range effect depends upon the localization of the variations and also on its nature. Regardless of the effect, all variations severely impaired the expression of α_IIbβ₃ integrin.

The expression of a functional α_IIbβ₃ integrin at the platelet surface requires: (1) the synthesis of each subunit; (2) their association and (3) their consecutive post-translational maturation. Hence the situation can be far more complex when considering the different aspects of the biosynthesis processing of proteins. Allosteric alterations identified (or suggested) here may affect conformations of the integrin complex not described by our reference structure - the initial structure obtained from a crystallized, bent, inactivated form of α_IIbβ₃. Interestingly, a study by Mitchell et al.⁴⁸ suggested that the α_IIb adopts a bent form early in its synthesis process. Finally functions of other proteins like chaperones or translocon potentially involved in the synthesis process could also be affected by α_IIbβ₃ structural alterations.

Although difficult to interpret in terms of pathogenicity our results showcased that aa substitutions identified in GT induced long-range alterations in the dynamics of the Calf-1 domain. To explore new potential pathogenic mechanisms, we are currently addressing the role of other variants in allosteric alterations of α_IIbβ₃ and particularly the role of neutral variants regarding the expression of the integrin α_IIbβ₃.