Fig 1.
Sequence alignment of rhodostomin and disintegrins containing an ARGDWN amino acid sequence.
Rhodostomin, cerastin, crotatroxin, durissin, lutosin, mojastin, tergeminin, and eristostatin are aligned, and the residues adjacent to the RGD motif and C-terminal regions are shown in boldface.
Table 1.
Inhibition of integrins αIIbβ3, αvβ3, and α5β1 by Rho and its 48ARGDWN mutants.
Table 2.
Inhibition of platelet aggregation integrins αIIbβ3, αvβ3, and α5β1 by Rho 48ARGDWN mutants.
Table 3.
Inhibition of integrins αIIbβ3, αvβ3, and α5β1 by Rho and its 48PRGDMP mutants.
Table 4.
Inhibition of platelet aggregation and the binding of fibrinogen to platelets by Rho ARGDWN mutants.
Fig 2.
2D 1H-15N HSQC spectra of Rho 48ARGDWN mutants at pH 6.
The peaks of Rho 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants are shown in red, blue, and green. Correlation peaks are labeled according to residues type and sequence number. The peaks connected by dotted lines correspond to Gln and Asn side chain NH2 group. In addition, the resonances of the side chains were labeled with the—sign. The chemical shift differences larger than 0.1 ppm are connected with the arrow, and we use the spectrum of 48ARGDWN-65PRYH mutant as the reference. The blue and green arrows represented the Rho 48ARGDWN-65PRNGLYG and 48ARGDWN-65PRNPWNG mutants.
Fig 3.
3D structures of Rho 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants.
Ribbon representation of 20 lowest-energy NMR structures of Rho 48ARGDWN-65PRYH (A), 48ARGDWN-65PRNGLYG (B), and 48ARGDWN-65PRNPWNG (C). Cartoon representation of the averaged structure of Rho 48ARGDWN-65PRYH (D), 48ARGDWN-65PRNGLYG (E) and 48ARGDWN-65PRNPWNG (F) mutant. The β-strands are shown in cyan. The structures are superposed on the main-chain atoms of the β-strands.
Table 5.
Summary of structural restraints and statistics for Rho 48ARGDWN mutants.
Fig 4.
Structural comparisons of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants.
(A) Ribbon representation of the averaged structures of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants are shown in red, blue, and green, respectively. Three two-stranded β sheets (13–14, 20–21, 33–34, 37–38, 43–45, and 55–57) were aligned. The RMSD deviations between 48ARGDWN-65PRYH and -65PRNGLYG mutants and between 48ARGDWN-65PRYH and -65PRNPWNG mutants are 0.681 Å and 0.423 Å. Structural alignments of the ten-residue RGD loop (residues 46 to 54) (B) and the C-terminal regions starting from the residue 64 to the C-terminal residue (C) of ARGDWN mutants are shown. The interactions between ARGDWN loop and their C-terminal regions of ARGDWN mutants are shown. The interactions of the W52 sidechain (yellow) with the H68 residue of 48ARGDWN-65PRYH mutant (D), with the L69 residue of 48ARGDWN-65PRNGLYG mutant (E), and with the N70 residue of 48ARGDWN-65PRNPWNG mutant (F) are shown. Interactions < 4Å are connected with the red broken lines. The sidechains of these C-terminal residues are colored in green.
Fig 5.
The docking of 48ARGDWN-65P, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants into integrin αIIbβ3.
The contact surface between the ARGDWN loop and integrin αIIbβ3 is shown in (A). The propeller domain of αIIb subunit and the A domain of β3 subunit are shown in purple and pink, respectively. The interacting residues are shown in the ball-and-stick representation, and hydrogen bonds are displayed by broken lines. The C-terminal 65PRNGLYG region of 48ARGDWN-65PRNGLYG (B) and the C-terminal 65PRNPWNG region of 48ARGDWN-65PRNPWNG (C) mutants are shown.
Table 6.
Summary of the interactions between Rho 48ARGDWN-65P, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants and integrin αIIbβ3.
Fig 6.
Structural comparison of rho and its 48ARGDWN-65PRYH mutant.
Ribbon representation of the ten-residue RGD loop (residues 46 to 54) (A) and all backbone (B) of the averaged structures of rho and its 48ARGDWN-65PRYH mutant are superimposed and shown in grey and red, respectively. The RMS deviation of the secondary structure backbone atom was 0.692 Å. The distances between Cα-to-Cα of the residues 52 and 68 of Rho and its 48ARGDWN-65PRYH mutant are 12.2 and 8.0 Å, respectively.
Table 7.
Comparison of the Cα(Ri)-Cα(Di+2), Cβ(Ri)-Cβ(Di+2), Cζ(Ri)-Cγ(Di+2), and Cα(Ri)-Cα(Xi+3) distances (Å) of integrin ligands.