Figure 1.
The initial structure of the β I domain of integrin αIIbβ3.
Amino acid residues from 109 to 354 of the β I domain of integrin αIIbβ3 (PDD: 3FCS) in Mg2+-Ca2+-Ca2+ state, and the ligand RGD coordinates were modeled by manual. (A) The complex structure of wild-type β I domain/RGD; (B) The local ligand metal-binding sites. The β I domain of integrin αIIbβ3 is shown in cartoon and colored green. The three residues Asp126, Asp127 and Ala252 are shown in stick and colored magenta. Ligand is shown in stick and colored cyan. N and O atoms are colored in blue and red, respectively. Magnesium is a green sphere, calcium ions are yellow, and crystal water molecules are red (Wat692, Wat756, Wat757, Wat761 in MIDAS site and Wat693, Wat694 in ADMIDAS site). Polar coordination between O atoms and metal ions are shown by dashed black lines. Figures are produced by PyMOL (www.pymol.org).
Figure 2.
Expression of WT and Mutant αIIbβ3 Integrins.
Immunofluorescent flow cytometry. HEK293T transfectants were labeled with AP3 (anti-β3), 7E3 (anti-β3), 10E5 (anti-αIIb), and LM609 (anti-αV). Thick and thin lines show labeling of the αIIbβ3 transfectant and the mock transfectant, respectively.
Figure 3.
Soluble Ligand Binding with PAC-1 and Fibrinogen.
Cells were incubated with (A) PAC-1 in the presence of 5 mM Ca2+ or 1 mM Mn2+ or (B) FITC-fibrinogen in the presence of 5 mM Ca2+ or 1 mM Mn2+ as indicated. Binding activities were determined by flow cytometry and expressed as described in Materials and Methods. Error bars are standard deviation (SD). An unpaired t-test with n=1000 for each group was conducted and showed to be statistically significant with a p-value <0.05 between WT and A252D for PAC-1 binding in Mn2+ conditions and fibrinogen binding in both Ca2+ and Mn2+ conditions, between WT and A252D/D126A as well as WT and A252D/D127A for PAC-1 and fibrinogen binding in both Ca2+ and Mn2+ conditions, and between A252D and A252D/D126A as well as A252D and A252D/D127A for PAC-1 and fibrinogen binding in Ca2+ conditions. However, insignificant results with a p-value >0.05 occurred between WT and A252D for PAC-1 binding in Ca2+ conditions and between A252D and A252D/D126A as well as A252D and A252D/D127A for PAC-1 and fibrinogen binding in Mn2+ conditions.
Figure 4.
A. Adhesion of HEK293T transfectants to surfaces coated with 20 µg/mL fibrinogen. The amount of bound cells was determined by measuring LDH activity as described in Materials and Methods. Data are representative of three independent experiments, each in triplicate. Error bars are SD. B. Quantification of the areas of adhering/spreading cells as described in Materials and Methods. C. DIC images of HEK293T transfectants after adhering to immobilized fibrinogen at 37°C. a: WT; b: A252D; c: A252D/D126A; d: A252D/D127A; e: A252D/D126A/D127A. The images are representatives of three independent experiments.
Figure 5.
The distributions of the eigenvalues on the first, second, and third principal components (PC).
The distributions of the eigenvalues on the PC1, PC2, and PC3 are demonstrated for Mg2+-Ca2+-Ca2+ [(a), (b), and (c)] and Mn2+-Mn2+-Mn2+ [(d), (e), and (f)] states.
Figure 6.
The view of the RGD binding site.
Final snapshots of the RGD binding site in (a) wild-type (WT) and mutant (b) A252D, (c) A252D/D126A, (d) A252D/D127A, (e) A252D/D126A/D127A from the molecular dynamics simulations in Mg2+-Ca2+-Ca2+ state. Ligand is shown in stick and colored cyan. N and O atoms involved in metal coordinating are colored in blue and red, respectively. Mg2+ is green, Ca2+ ions are yellow, and coordinating water molecules are red. Polar coordination between O atoms and metal ions are shown by dashed black lines.