biotinylated human upar antibody  (R&D Systems)

Journal: Biochemistry

Article Title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

doi: 10.1021/acs.biochem.6b01039

Figure Lengend Snippet: (a) Three-dimensional structure of the uPAR·uPAATF complex (PDB entry 2FD6). uPAR is shown as a gray solvent-accessible surface. Residues whose mutations to alanine result in changes in binding affinity of ≥1 kcal mol−1 are colored red, while those that lead to changes between 0.5 and 1 kcal mol−1 are colored purple. uPAATF is shown as an orange cartoon. (b) Three-dimensional structure of uPAR in complex with the AE-157 peptide (PDB entry 1YWH). The peptide is shown as a cyan cartoon, and side chains that come in contact with uPAR are shown as capped sticks. uPAR is rendered and color-coded in a manner similar to that of panel a.

Article Snippet: Following incubation for 30 min and subsequent washing steps, the human uPAR biotinylated antibody (1:3000 dilution of 0.2 mg mL −1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% BSA was added to the wells (100 μ L/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Solvent, Binding Assay

Journal: Biochemistry

Article Title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

doi: 10.1021/acs.biochem.6b01039

Figure Lengend Snippet: Mode of binding of 12 (IPR-1175) upon the central ligand binding cavity of suPARcc. suPARcc is shown as a gray solvent-accessible surface. 12 (IPR-1175) is shown as capped sticks. Atoms are color-coded by atom type with N, C, O, Cl, and F colored blue, yellow, red, green, and light blue, respectively. The 2Fo – Fc electron density map of 12 (IPR-1175) at 1σ is colored blue. (b) Crystal structure of 12 (IPR-1175) bound to uPAR except that uPAR is shown as capped sticks to highly hot-spot residues located within the uPAR cavity. Hot spots are shown as red capped sticks. Arg-53, which is not considered a hot spot, is colored green. (c) Crystal structure of the uPAR·12 (IPR-1175) complex superimposed on the structure of the uPAR·uPAATF complex to illustrate the overlap between hot spots on uPA and substituents on 12 (IPR-1175).

Article Snippet: Following incubation for 30 min and subsequent washing steps, the human uPAR biotinylated antibody (1:3000 dilution of 0.2 mg mL −1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% BSA was added to the wells (100 μ L/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Ligand Binding Assay, Solvent

Journal: Biochemistry

Article Title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

doi: 10.1021/acs.biochem.6b01039

Figure Lengend Snippet: Mode of binding of 3 (IPR-737) upon the central ligand binding cavity of suPARcc. suPARcc is shown as a gray solvent-accessible surface. 3 (IPR-737) is shown as capped sticks. Atoms are color-coded by atom type with N, C, O, Cl, and F colored blue, yellow, red, green, and light blue, respectively. A 2Fo – Fc electron density map of 3 (IPR-737) at 1σ is colored blue. (b) Crystal structure of 3 (IPR-737) bound to uPAR except that uPAR is shown as capped sticks to highly hot-spot residues located within the uPAR cavity. Hot spots are shown as red capped sticks. (c) Crystal structure of the uPAR·3 (IPR-737) complex superimposed on the structure of the uPAR·uPAATF complex to illustrate the overlap between hot spots on uPA and substituents on 3 (IPR-737).

Article Snippet: Following incubation for 30 min and subsequent washing steps, the human uPAR biotinylated antibody (1:3000 dilution of 0.2 mg mL −1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% BSA was added to the wells (100 μ L/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Binding Assay, Ligand Binding Assay, Solvent

Journal: Biochemistry

Article Title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

doi: 10.1021/acs.biochem.6b01039

Figure Lengend Snippet: (a) Plot of the MM-GBSA free energy vs inhibition constant Ki that was measured using the fluorescence polarization assay. The correlation coefficients of all 12 selected derivatives are as follows: r = 0.48, ρ = 0.41, and τ = 0.29. (b) Decomposition of the free energy of binding for uPA, 12 (IPR-1175), and 3 (IPR-737). The decomposition energy consists of the interaction energy between the ligand and each residue on uPAR determined for a collection of snapshots that were obtained from molecular dynamics simulations. (c) Decomposition energies for a select number of hot-spot and non-hot-spot residues for a set of 1 (IPR-1110) derivatives.

Article Snippet: Following incubation for 30 min and subsequent washing steps, the human uPAR biotinylated antibody (1:3000 dilution of 0.2 mg mL −1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% BSA was added to the wells (100 μ L/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: Inhibition, Fluorescence, Binding Assay, Residue

Journal: Biochemistry

Article Title: Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein–Protein Interaction

doi: 10.1021/acs.biochem.6b01039

Figure Lengend Snippet: (a) Dynamic cross-correlation matrix (DCCM) cross section of 3 and select derivatives of 1 with uPAR. (b) DCCM of uPAR in the uPAR·uPA complex, colored using the same scheme used for panel a. (c) Pearson’s correlation between the cross-correlation of uPAR·3 and uPAR·12 complexes and the cross-correlation of individual residues of uPAR in the uPAR·uPA complex. Stereoviews of three-dimensional structures of uPAR in the (d) uPAR·3 and (e) uPAR·12 complexes, colored on the basis of the correlation coefficient in panel c from positive correlation (blue) to no correlation (white) to negative correlation (red).

Article Snippet: Following incubation for 30 min and subsequent washing steps, the human uPAR biotinylated antibody (1:3000 dilution of 0.2 mg mL −1 BAF807, R&D Systems, Minneapolis, MN) in PBS containing 1% BSA was added to the wells (100 μ L/well) and incubated for 1 h to allow for the detection of bound uPAR.

Techniques: