Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

Abstract

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser-Asp-Val (SDV) sequence and integrin alpha v beta 3. P11 (HSDVHK) is a novel antagonistic peptide of integrin alpha v beta 3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin-binding site of integrin alpha v beta 3. The Arg-Gly-Asp (RGD)-binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin-vitronectin. P11 showed a strong antagonism against alpha v beta 3-GRGDSP interaction with an IC(50) value of 25.72+/-3.34 nM, whereas the value of GRGDSP peptide was 1968.73+/-444.32 nM. The binding-free energies calculated from the docking simulations for each P11 and RGD peptide were -3.99 and -3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5-fold lower K(i) value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in alpha v beta 3. The analyzed docked poses suggest that a divalent metal-ion coordination was a common driving force for the formation of both SDV/alpha v beta 3 and RGD/alpha v beta 3 complexes. This is the first report on the specific recognition of the RGD-binding site of alpha v beta 3 by a non-RGD containing peptide using a computer-assisted proteomic approach.

Pharmacoproteomic Analysis of a Novel Cell-permeable Peptide Inhibitor of Tumor-induced Angiogenesis

“…P11 has been shown to inhibit the integrin ␣ v ␤ 3 -vitronectin interaction in a dose-dependent manner (21). The SDV sequence of P11 is able to recognize the vitronectin-binding site (RGD-binding site) of integrin ␣ v ␤ 3 in a site-specific manner (22). To determine the antiangiogenic mechanism of P11 as an antagonist of integrin ␣ v ␤ 3 , we first examined the effect of P11 on endothelial cell proliferation.…”

“…In our previous study, P11, a novel peptide sequence containing Ser-Asp-Val (SDV), was shown to specifically bind to integrin ␣ v ␤ 3 instead of the RGD sequence, a common binding motif of the integrin receptor (22). The SDV sequence is a type I PDZ-binding domain (S/T-X-V) that can recognize PSD-95 (63).…”

Complement Factor H, Vitronectin, and Opticin Are Tyrosine-Sulfated Proteins of the Retinal Pigment Epithelium

“…Since this size is similar to the heavy chain of the antibody, which is also tyrosine-sulfated [52], opticin was immunoprecipitated using anti-opticin antibody and eluted under non-reducing conditions, so that the tyrosine-sulfated heavy chain of the antibody would not mask the opticin isoform. The anti-opticin antibody was successful in bringing down the 55 kD monomeric opticin band as well as multiple aggregated opticin bands of sizes 55–75 kD, with the 75 kD band being the most abundant (asterisks, Figure 4A).…”

“…These two residues are close to the ‘RGD’ cell attachment site on the protein, which resides between residues 64–66. The RGD sites on vitronectin have been previously shown to bind integrin receptor αvβ3 and αvβ5 [52], [65]. However, for this to occur, the ‘RGD’ site must be exposed to the surface, which can be influenced by type of surrounding residues.…”

Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides

“…Kang and co-workers used integrin microarrays to select peptides that inhibit angiogenesis. 345,346 They first used an α v β 3 microarray and a PS-SPCL to screen for peptides that interfered with the α v β 3 –vitronectin interaction. Fluorescently labeled vitronectin was mixed with the peptide library before addition to the microarray.…”