Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-65PRYH and 48ARGDWN-65PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48ARGDWN-65PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbβ3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-π interaction with the β3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.

Introduction RGD-containing disintegrins are potent integrin inhibitors that were found in snake venoms [1][2][3][4]. They are classified into small, medium, long, and dimeric disintegrins based on their size and the number of disulfide bonds [5]. Short disintegrins are composed of 41 to 51 residues and four disulfide bonds; medium disintegrins contain approximately 70 amino acids and six disulfide bonds; long disintegrins include a polypeptide with approximately 84 residues cross-linked by seven disulfide bonds; and homo-and hetero-dimeric disintegrins contain each subunit of approximately 67 residues with a total of ten disulfide bonds involved in the formation of four intrachain disulfides and two interchain disulfides [6]. A common structural feature of RGD-containing disintegrins is the presence of a solvent-exposed RGD tripeptide, which is crucial to the recognition of integrins [7]. The pairing of cysteine residues in disintegrins play an important role in exposing the RGD binding motif that mediates inhibition of platelet aggregation, neutrophil or endothelial cell function [1][2][3][4][5][6][7]. Disintegrins are therefore used to develop anti-platelets agents and anti-angiogenesis inhibitors for cancer [1][2][3][4][5][6].
Many studies have shown that the residues flanking the RGD motif and in the C-terminal region of disintegrins affect their integrins binding specificities and affinities [8][9][10][11][12][13][14][15]. For example, disintegrins with an ARGDW sequence exhibit a higher affinity for binding with integrin αIIbβ3, whereas disintegrins with an ARGDN sequence preferentially bind with integrins αvβ3 and α5β1 [10]. The amino acid sequences of RGD loop of rhodostomin (Rho) was mutated from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in the inhibition of integrin αIIbβ3 [12]. Replacing the N-terminal alanine with the proline of the RGD motif of elagantin (a disintegrin with an ARGDMP sequence) diminishes its ability to bind to integrin α5β1 [13]. The N-terminal proline residue adjacent to the RGD motif of Rho affects its function and dynamics [14]. Deletion and mutagenesis studies on echistatin have demonstrated that its C-terminal tail is important for its activity in inhibiting platelet aggregation [11,15].
Many functional studies showed that the C-terminal tails of disintegrins act with the RGD loop to regulate integrins recognition [8,11,[16][17][18][19][20][21][22]. For example, Marcinkiewicz et al. reported that the C-terminal region of echistatin supports integrin binding and plays a crucial role in the expression of ligand-induced binding site (LIBS) epitope and in the conformational changes of the integrins [11]. The C-terminal tail 66 RWN residues of trimestatin are positioned close to the C-terminal side of the RGD loop and act as a secondary determinant of integrinbinding potency [18]. In particular, eristostatin requires an ARGDW motif and an intact Cterminus (NPWNG) to interact with both platelets and melanoma cells [19]. Eristostatin and bitistatin contain an ARGDWN motif with different C-terminal tails, and eristostatin exhibits a higher affinity to resting platelets [20,23]. However, the structural basis and mechanism underlying how integrins are recognized by the C-terminus and RGD loop of disintegrins are unclear.
To examine how the C-terminus interacts with the RGD loop to recognize integrin αIIbβ3, we analyzed disintegrins containing an ARGDWN loop and found that they mainly exhibited C-termini with NGLYG and NPWNG amino acid sequences (Fig 1). Therefore, we used Rho as the model protein to study the effects of the ARGDWN/PRGDMP loops and C-terminal regions on the structure-activity relationships of disintegrin. Rho is obtained from Calloselasma rhodostoma venom and belongs to the disintegrin family [24]. It consists of 68 amino acids, including 12 residues of cysteine and a PRGDMP sequence at positions 48 to 53. We have demonstrated that Rho expressed in Pichia pastoris has the same function and structure as the native protein [25,26]. In this study, we expressed Rho containing an 48 ARGDWN or 48 PRGDMP loop with different C-terminal sequences in P. pastoris, determined their activity in the inhibition of integrins, and used nuclear magnetic resonance (NMR) spectroscopy to compare their structural differences. We also docked these mutants into integrin αIIbβ3 and analyzed their interactions. The results demonstrated that the RGD loop and C-terminus of medium disintegrins interact with each other, resulting in structural and functional differences relevant to integrin binding.

Mass spectrometric measurements
The molecular weights of proteins were confirmed using an LTQ Orbitrap XL mass spectrometer equipped with an electrospray ionization source (Thermo Fisher Scientific). The protein solutions (1-10 μM in 50% methanol with 0.1% formic acid) were infused into the mass spectrometer by using a syringe pump at a flow rate of 3 μL/min to acquire full scan mass spectra. The electrospray voltage at the spraying needle was optimized at 4000 V. The molecular weights of proteins were calculated by computer software Xcalibur that was provided by the Thermo Fisher Scientific.

Cell adhesion assay
The adhesions of CHO-αIIbβ3 cells to fibrinogen, CHO-αvβ3 cells to fibrinogen, and K562 cells to fibronectin were used to determine the inhibitory activities of Rho mutants to integrins αIIbβ3, αvβ3, and α5β1. They were conducted according to previously described protocols [14,27].

Preparation of human platelets
Platelets were collected using 0.15 vol/vol acid-citrate dextrose (ACD) containing 85 mM trisodium citrate, 2% dextrose and 65 mM citric acid as the anticoagulant and washed using a modification of a previously described method [29]. 12 mL of blood was centrifuged at 150 × g for 10 min at room temperature (RT). The buffy coat and the red blood layers were discarded to avoid the contaminants. The remaining 5 ml of platelet-rich plasma (PRP) layer was acidified to pH 6.5 with 5 ml of ACD and then added 1 μL of 10 mM prostaglandin E1 (PGE1). Platelets were pelleted by centrifugation at 750 g for 10 min at room temperature (RT), and the supernatant was removed. The platelet pellet was gently re-suspended in 5 mL of 130 mM NaCl, 3 mM KCl, 10 mM trisodium citrate, 9 mM NaHCO 3 , 6 mM dextrose, 0.9 mM MgCl 2 , 0.81 mM KH 2 PO 4 , and 10 mM Tris (JNL) buffer at pH 7.4. Platelets were counted using a XT-1800-Hematology-Analyzer and were adjusted to 1×10 8 per ml. Platelets were allowed to stand at RT for 45 min to let PGE1 dissipate. 20 μL of 18 mM calcium chloride was immediately added into 2 mL of platelet solution before the fibrinogen binding experiment.

Fibrinogen binding assay
The fibrinogen (Fg) binding assay was accomplished using a modification of a previously described method [29]. Rho and its mutants (40-2000 nM), which were used as inhibitors, were added to 5 μL of 2.5 mg/mL Oregon Green 488-labeled fibrinogen (Invitrogen, UK). 20 μL aliquots of washed platelet suspension were then added and incubated for 30 min before the addition of 10 μM ADP. The resulting platelet solutions were incubated at RT for a further 30 min. The reaction was stopped by addition of 1 mL ice-cold buffer. The binding of Fg to platelets was detected using a flow cytometry. Data acquisition and analysis were performed with the Cell Quest program. Platelet populations were gated for the analysis, and the histograms of mean fluorescence were generated for each sample. Statistical analysis was performed on the geometric scale. All experiments were run in duplicate, and the reported IC 50 values are the average of at least three separate experiments.

Platelet aggregation assay
The inhibition of platelet aggregation by Rho mutants was accomplished by following previously described protocols [14,27].

Molecular docking
The docking of Rho mutants to integrin αIIbβ3 was performed on the HADDOCK webserver by using hydrogen bond and distance restraints as described previously [33]. The starting structures for the docking were NMR structures of Rho mutants and integrin αIIbβ3 (PDB code 3ZE2) [34]. The interaction restraints were derived from the X-ray structure of integrin αIIbβ3 in complex with a GRGDSP peptide by using CCP4i software (http://structure.usc.edu/ccp4/). The selected structure cluster for the analysis was based on the lowest Z-score without any restraint violations. Hydrogen bonds and salt bridges were analyzed using PISA software (http://www.ebi.ac.uk/msd-srv/prot_int/). Cation-π interactions and non-bonded contacts were determined using CaPTURE (http://capture.caltech.edu/) and CCP4i, respectively [35].
Protein data bank accession number and nuclear magnetic resonance assignment

Protein expression and purification of rhodostomin mutants
Rho mutants were expressed in P. pastoris X33 strain by using the pPICZαA vector. Recombinant Rho mutants proteins were purified to homogeneity by Ni 2+ -chelating chromatography and C18 reversed-phase HPLC. According to SDS-polyacrylamide gel electrophoresis analysis (data not shown), the purified Rho mutants proteins were homogenous. The final yields of unlabelled Rho mutants produced in P. pastoris were 10 to 25 mg/L, and the final yields of 15 N-labeled Rho mutants were 5 to 15 mg/L. Mass spectrometry was used to determine the molecular weights of recombinant Rho mutants. Mass spectrometry indicated that the experimental molecular weights deviated less than 1 Da from the theoretical values, which were calculated by assuming that all cysteines formed disulfide bonds in Rho mutants.  Table A in S1 File).
We expressed a series of Rho C-terminal mutants to confirm their effects on inhibiting integrins ( Table 2) We also expressed Rho mutants containing a 48 PRGDMP sequence with different C-terminal tails, including 48 PRGDMP-65 PR, 48 PRGDMP-65 PRYH, 48 PRGDMP-65 PRNGLYG, and 48 PRGDMP-65 PRNPWNG mutants, to examine the roles of the C-terminal regions (Table 3). Their affinity differences in inhibiting integrins αIIbβ3, αvβ3, and α5β1 were ranged from 1.0 to 11.4-, 1.0 to 1.8-, and 0.9 to 2.3-folds. These results indicated that the effects of C-terminal regions on the change of their relative binding affinity to integrins was αIIbβ3 > α5β1 ! αvβ3. In contrast, the 48 PRGDMP-65 PRNPWNG mutant did not exhibit any integrin selectivity and inhibited integrins αIIbβ3, αvβ3, and α5β1 with IC 50 values of 235.2, 40.7, and 260 nM, respectively. These findings revealed that the 48 ARGDWN sequence selectively inhibited integrin αIIbβ3. We also found that the incorporation of C-terminal NPWNG sequence with ARGDWN loop increased the inhibitory activity against integrin αIIbβ3.  (Table 4). These results showed that the length of the C-terminus and the R66 residue of Rho with an 48 ARGDWN loop sequence are essential for interacting with platelets integrin αIIbβ3. We also expressed Rho mutants containing a 48 PRGDMP sequence with different C-terminal tails to examine the C-terminal effect on inhibiting platelet aggregation ( Table B in S1 File). The IC 50 values of 48 PRGDMP-65 PR, 48 PRGDMP-65 PRYH, 48 PRGDMP-65 PRNGLYG, and 48 PRGDMP-65 PRNPWNG proteins were 155.2, 83.2, 96.9, and 130.9 nM, respectively. These results also showed that the length and amino acid contents of the C-terminus in Rho with a 48 PRGDMP loop sequence may play a critical role in interacting with platelets integrin αIIbβ3. ARGDWN-65 P mutant exhibited 3.2-fold decrease in inhibiting the association between washed platelet and soluble fibrinogen in comparison with that of 48 ARGDWN-65 PRNPWNG mutant. These results were consistent with the results of platelet aggregation that the length of the C-terminus and the R66 residue of 48 ARGDWN mutants are essential for interacting with platelets integrin αIIbβ3. In contrast to the result of the adhesion of cell-expressing integrin αIIbβ3 to immobilized fibrinogen, 48 ARGDWN-65 P mutant exhibited significant effect with 23.1-fold decrease in activity.
According to the NOE spectra, the conformational differences were found in the C-terminal regions and their interactions with the ARGDWN loop ( Figure C in S1 File). The NPWN residues of the 48 ARGDWN-65 PRNPWNG mutant formed a β-turn structure, which was reflected by the NOEs between H α of N67 and H N of N70 and between H α of P68 and H N of N70. In contrast, no turn structure was identified from the C-   Superimposing 3D structures of these mutants demonstrated that their overall structures were similar, except for the C-terminal regions and their interactions with the 48 ARGDWN loop (Fig 4). The structural analysis also indicated that their 48 ARGDWN loop exhibited similar conformations (Fig 4B). The C-terminal regions of these mutants exhibited distinct   The roles of RGD loop and C-terminus of medium disintegrins conformations: the YH residues of the 48 ARGDWN-65 PRYH mutant had an extended structure; the NGLYG residues of the 48 ARGDWN-65 PRNGLYG mutant had a turn-like structure; and the NPWN residues of the 48 ARGDWN-65 PRNPWNG mutant formed a β-turn structure ( Fig 4C). The interactions between the ARGDWN loop and C-terminal regions were extremely different. Our analysis demonstrated that the W52 sidechain of the 48 ARGDWN-65 PRYH, 48 ARGDWN-65 PRNGLYG, and 48 ARGDWN-65 PRNPWNG mutants mainly interacted with H68 of -65 PRYH (Fig 4D), with L69 of -65 PRNGLYG (Fig 4E), and with N70 of -65 PRNPWNG (Fig 4F), respectively. In addition, the A48 sidechain of the 48 ARGDWN-65 PRNPWNG mutant interacted with the N70 residue of the C-terminal region, and this interaction was not found in two other mutants. These structural differences may be correlated with their functional differences.

ARGDWN-65 PRNPWNG mutant complexes
The docking of 48 ARGDWN-65 P, 48 ARGDWN-65 PRNGLYG, and 48 ARGDWN-65 PRNPWNG mutants into integrin αIIbβ3 was used to simulate their interactions with integrin αIIbβ3. The The roles of RGD loop and C-terminus of medium disintegrins models of these integrin αIIbβ3 complexes were built using the HADDOCK webserver [33]. The distance and hydrogen bond restraints were derived from the X-ray structure of integrin αIIbβ3 complexed with a GRGDSP hexapeptide (PDB code 3ZE2), including eight key interactions between integrin and the R and D residues (Table C in S1 File). Specifically, the R residue formed a salt bridge with the D224, hydrogen bonds with the Y189 and S225 residues, and a cation-π interaction with the F231 of the αIIb subunit. The carboxylate oxygen of the D residue contacted a Mn 2+ ion and formed hydrogen bonds with the S123 residues of the β3 subunit. The other carboxyl oxygen of the D residue formed hydrogen bonds with the Y122 and N215 residues of the β3 subunit, and the backbone amide of the D residue formed a hydrogen bond with the R216 residue of subunit β3.
Using these restraints, we docked Rho ARGDWN mutants to integrin αIIbβ3. The structure cluster was selected based on the lowest Z-score without restraint violations. The Z-score values of the 48 ARGDWN-65 P, 48 ARGDWN-65 PRNGLYG, and 48 ARGDWN-65 PRNPWNG mutants were -1, -1.2, and -1, and their electrostatic energies were -515.9, -569.5, and -630.7 kcal/mol, respectively (Table D in S1 File). This was consistent with the effects of cell adhesion data on integrin αIIbβ3 that 48 ARGDWN-65 P and 48 ARGDWN-65 PRNPWNG mutants exhibited the lowest and highest inhibitory activities. The resulting structures showed that Rho mutants fitted into a crevice between the propeller domain of the αIIb subunit and the βA domain of the β3 subunit on the αIIbβ3 headpiece. The analysis showed that the docking of these mutants into integrin αIIbβ3 resulted in the same numbers of contacts for the 48 ARGDWN loop ( Table 6). The key contacts included seven hydrogen bonds and two salt bridges between the R and D residues of the RGD motif and integrin. In particular, the contacts of the hydrogen bond and salt bridge between the R49 residue of Rho mutants and the Y189 and D224 residues of the αIIb subunit, and the hydrogen bond between the D51 residue of Rho mutants and the Y122, S123, N215, and R216 residues of the β3 subunit were exhibited by all the mutants (Fig 5A). The major differences between the mutants were the interactions between integrin αIIbβ3 and the C-terminal regions of the Rho mutants ( Table 6). The C-terminal region of the 48 ARGDWN-65 P deletion mutant did not exhibit any interaction with integrin αIIbβ3 (Table 6). In contrast, the C-terminal regions of the 48 ARGDWN-65 PRNGLYG and 48 ARGDWN-65 PRNPWNG mutants extensively interacted with integrin αIIbβ3 (Table 6). For example, the C-terminal region of the G71 residue of the 48 ARGDWN-65 PRNGLYG mutant formed a hydrogen bond with the V156 residue of the αIIb subunit (Fig 5B and  Table 6). The W69 and N70 residues of 48 ARGDWN-65 PRNPWNG exhibited cation-π interaction with the K125 residue of the β3 subunit and a hydrogen bond with the D159 residue of the αIIb subunit ( Fig 5C and Table 6). In contrast to 48 ARGDWN-65 P mutant, the R66 residue of 48 ARGDWN-65 PRNGLYG and 48 ARGDWN-65 PRNPWNG mutants interacted with the D159 residue of the αIIb subunit. These results suggested that the contents of the C-terminal regions in disintegrins are critical to their abilities to bind to integrin αIIbβ3.

Discussion
Many studies have shown that alternations in the RGD loop and C-terminal region of disintegrins affect their binding specificities and affinities [8][9][10][11][12][13][14][15][16][17][18][19]. In this study, we find that the sequence contents of the RGD loop and C-terminus of disintegrins mutually affected their conformations, resulting in functional and structural differences in integrin binding. We demonstrated that Rho mutants containing a 48 ARGDWN-65 PRNPWNG sequence exhibited the highest selectivity in inhibiting integrin αIIbβ3-mediated cell adhesion. Cell adhesion analysis also indicated that the C-terminal region of Rho was highly sensitive to integrin αIIbβ3. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbβ3 of platelets bound differently to immobilized and soluble fibrinogen. The results of platelet aggregation integrin and αIIbβ3-mediated cell adhesion showed that the R66 and Y67 residues may play important roles in inhibiting the binding of platelet to soluble fibrinogen and the adhesion of integrin αIIbβ3 to immobilized fibrinogen, respectively. NMR structural analysis of 48 ARGDWN-65 PRYH, 48 ARGDWN-65 PRNGLYG, and 48 ARGDWN-65 PRNPWNG mutants showed that their C-terminal regions The roles of RGD loop and C-terminus of medium disintegrins exhibited distinct conformations. Molecular docking results suggest that the sequence contents and the length of the C-terminal regions in disintegrins are critical to their ability to bind to integrin αIIbβ3. We provide the first structural evidence that the diverse RGD loop and C-terminus of medium disintegrins interact to regulate their conformations, resulting in functional differences in integrin binding. The structural analysis of wild-type Rho and its 48 ARGDWN mutants also showed that a conformational difference existed in the 3D conformation of the RGD loop ( Fig 6A). Many studies have demonstrated that a key feature of integrin αIIbβ3 antagonists is the presence of an anionic carboxy-terminal (CO 2 -) separated by a spatial chemical moiety and a certain distance from the cationic basic amino-terminal of benzamidine, piperidine, and guanidine [36]. The distance between the anionic (D) and cationic (R) terminals is crucial to the optimal binding affinity and specificity for various integrins. Specifically, the distances between the R and D residues of RGD-containing peptides can be optimally designed for the selective recognition of integrins αIIbβ3, αvβ3, and α5β1 [37]. Therefore, we analyzed the distances between Cα-to-Cα, Cβ-to-Cβ, and Cz-to-Cγ of the R(i) and D(i+2) residues, and between Cα-to-Cα of the R(i) and X(i+3) residues of Rho, its mutants, and RGD-containing peptides (Table 7). We found that the distance between the Cα-to-Cα of the R(i) and X(i+3) residues was correlated with their integrin specificity. The Cα-to-Cα distances of the R(i) and X(i+3) residues in eptifibatide, an integrin αIIbβ3 antagonist, and in cilengitide, an integrin αvβ3/αvβ5 antagonist, were 7.6 and 5.4 Å, respectively. The average Cα-to-Cα distances of the R(i) and W/M(i+3) residues in Rho 48 ARGDWN mutants and Rho with a 48 PRGDMP sequence were 7.3 to 7.6 Å and 6.5 Å, respectively. These results indicated that the Cα-to-Cα distances of the R(i) and X(i+3) residues of the integrin αIIbβ3-specific antagonist were larger than that of the integrin αvβ3 antagonist. This demonstrated that the W52 residue increased the Cα-to-Cα distance between R(i) and W(i+3) of the 48 ARGDWN motif, resulting in its selectivity to integrin αIIbβ3. Our results were consistent with the previous hypothesis that integrin αIIbβ3-specific disintegrin prefers a larger Cα(i)-to-Cα(i+3) distance in its RGDX motif [8].
Many studies have shown that the C-terminal tails of disintegrins are located in the proximity of the RGD loop, the integrin-binding loop, and that the C-terminal regions of disintegrins play synergistic roles in interacting with RGD-binding integrins [16,18,20,21,25]. For example, C-terminal W67 of flavoridin with an 48 ARGDFP motif is close to D55 [21], C-terminal Y67 of Rho with a 48 PRGDMP motif is close to D55 [25], and C-terminal W67 of trimestatin with a 48 ARGDNP motif is close to P53 [18]. The structural analysis of wild-type Rho and its 48 ARGDWN mutants also showed that a conformational difference existed in their RGD loop and C-terminal region (Fig 6B). In contrast to that of Rho, structural analyses of the Rho 48 ARGDWN-65 PRYH mutant indicated that C-terminal H68 is close to W52. C-terminal L69 of the 48 ARGDWN-65 PRNGLYG mutant is close to W52, and the C-terminal N70 residue of the 48 ARGDWN-65 PRNGWNG mutant is close to W52 and A48. We also found that the 67 NPWN region of the 48 ARGDWN-65 PRNPWNG mutant formed a type I β-turn, which was not found in other C-terminal mutants. These results suggest that the sequence contents of the C-terminal region and RGD loop of disintegrins are important for their 3D conformation and mutual interactions. These structural differences may be correlated with their functional differences. Table 7. Comparison of the C α (R i )-C α (D i+2 ), C β (R i )-C β (D i+2 ), C ζ (R i )-C γ (D i+2 ), and C α (R i )-C α (X i+3 ) distances (Å) of integrin ligands. The roles of RGD loop and C-terminus of medium disintegrins Integrins are known for their ability to bind multiple ligands due to flexibility in their binding sites. Many studies showed that integrin αIIbβ3 adhesion on fibrinogen is mediated by recognition sequences RGDF (Aα95-98), RGDS (Aα572-575), and HHLGGAKQAGDV (γ 400-411) of fibrinogen [38][39][40][41]. In particular, different recognition sites of soluble and immobilized fibrinogen are used for their binding to integrin αIIbβ3 [38,39]. For example, integrin αIIbβ3 binds to soluble fibrinogen mainly through HHLGGAKQAGDV (γ 400-411). Integrin αIIbβ3 binds to immobilized fibrinogen through not only HHLGGAKQAGDV (γ 400-411) but also RGDF (Aα95-98). In contrast to integrin αvβ3 adhesion on fibrinogen, it is only mediated by the carboxyl-terminal RGDS site of the Aα chain [4141]. Our findings revealed that Rho 48 ARGDWN mutants selectively inhibited integrin αIIbβ3 to immobilized and soluble fibrinogen, and the incorporation of C-terminal NPWNG increased its inhibitory activity to immobilized fibrinogen. However, it is likely that the specificity of Rho ARGDWN mutants with Cterminal PRNPWNG sequence towards αIIbβ3 antagonism could be due to better competition with not only RGD but also the γ-chain ligands as well. Although functional and structural differences in ARGDWN mutants and their integrin αIIbβ3 complexes were found from our study, it is uncertain that these interactions may take place in vivo and are affected by the ionic milieu. The effect of recognition by the inside-out signaling on integrin cannot be also excluded as well.
In conclusion, our functional and structural analyses demonstrated that the RGD loop and C-terminus of rhodostomin mutants interact with each other. The amino acid sequences of the RGD loop and C-terminal regions in medium disintegrins are important for their interactions and abilities to the binding of integrin αIIbβ3 to immobilized and soluble fibrinogen. These findings provide new insights into the structure-based drug design of integrin αIIbβ3 antagonist by using the disintegrin scaffold, and they serve as a basis for exploring the structure-function relationships of RGD-binding integrins and their ligands.
Supporting information S1 File.