Journal: Nature Communications

Article Title: Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection

doi: 10.1038/s41467-021-21972-0

Figure Lengend Snippet: a Protein–protein docking analysis of a homology model for the TMPRSS2 extracellular fragment (green, PDB 1z8g) and α 1 AT (gray, PDB 3cwm) and computationally calculated binding free energy (ΔG calc ) of the complex. b Detailed view on α 1 AT-TMPRSS2 binding interface. The sidechains of α 1 AT (gray) residues are represented with sticks, while sidechains of TMPRSS2 (green) are shown with balls and sticks. Hydrogen atoms are omitted for clarity, carbon, oxygen, nitrogen or sulfur atoms of amino acid side chains depicted in light blue, red, dark blue or yellow, respectively. The hydrophobic patch near the TMPRSS2 catalytic triad is highlighted with a green transparent surface. c α 1 AT inhibits cell-associated TMPRSS2 activity. HEK293T cells were transfected with a TMPRSS2 expression plasmid and treated with Prolastin (α 1 AT, blue), camostat mesylate (CM, gray) or E-64d (green) followed by incubation with the fluorogenic TMRPSS2 protease substrate BOC-Gln-Ala-Arg-AMC. Graph shows the relative area under the curve analysis of fluorescence intensities over 2 h that were corrected by values for mock-transfected HEK293T cells. d α 1 AT inhibits recombinant TMPRSS2 enzyme activity. Recombinant human TMPRSS2 was mixed with Prolastin (α 1 AT, blue) or CM (gray) prior to addition of fluorogenic TMPRSS2 protease substrate BOC-Gln-Ala-Arg-AMC, graph shows relative fluorescence intensities after 3 h of incubation. The mean ± SEM of n = 2 ( c ) or n = 3 ( d ) independent experiments in biological duplicates are shown (ordinary one-way ANOVA with Dunett´s multiple comparison test). Source data are provided as a Source data file.

Article Snippet: For assessing the activity of recombinant human TMPRSS2, 25 µl of serially diluted Prolastin or inhibitor control in assay buffer (50 mM Tris-HCL, 0.154 mM NaCl pH 8.0) were incubated with 25 µl of recombinant TMPRSS2 enzyme for 10 min at 37 °C, followed by addition of 20 µM BOC-Gln-Ala-Arg-AMC protease substrate.

Techniques: Binding Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Fluorescence, Recombinant