(E) CPMG spectra of methionine as a negative control. The binding of the μPlm (green) leads to a reduction of signals and also a shift of the signal at 7.25, 6.94, and 6.75 ppm, which indicates strong binding of YO-2 to μPlm. (D) CPMG spectra of YO-2 as a positive control. The ligand signals in the presence of μPlm (green) at 2.8 to 2.75 ppm are attenuated, which indicates binding. TXA binding to μPlm is indicated by the reduction in peak intensity in the presence of μPlm. TXA signals are compared in the presence (green) and absence (red) of μPlm. (B) Purcell-Meiboom-Gill (CPMG) spectra confirm binding of TXA to μPlm. The bottom line (red) shows the STD spectrum in the absence of μPlm there are no signals, as expected. The middle line (green) shows the STD signals of TXA in the presence of μPlm positive STD signal at 2.6 and 2.58 ppm of TXA with an intensity of 5% is indicative of TXA binding to μPlm. The top line (blue) shows the reference spectrum of TXA alone. (A) Saturation-transfer difference (STD) spectra reveal binding of TXA to μPlm. Inhibitor binding to Plm determined by NMR. Taken together, these data suggest that F587 plays a substantive and important role in the inhibitor interaction. The IC 50 for the double mutant (F587A/K607A) is similar to that of F587A. The results reveal that although the IC 50 for K607A is marginally lower than the wild-type, that for the F587A variant is much higher (four- to fivefold) than the wild-type. To study the role of F587 and K607 in the interaction with the YO compounds, we determined the IC 50 using F587A and K607A single and double mutants ( Table 1). Binding of YO-2 does not have an impact on the geometry of the catalytic triad residues (H603, D646, and S741) because they remain in their catalytic conformations ( Figure 1B supplemental Figure 1). The hydrophobic aliphatic octylamide does not have extensive interactions with the acidic S2′ site instead, it points away from the protease. Of note, the inhibitor forms particularly extensive interactions at the S3′ subsite where the tyrosine moiety forms a hydrogen bond with K607, and the pyridine moiety forms an imperfect face-to-face π stacking with the benzyl side chain of F587. The remainder of the inhibitor occupies the S1′-S3′ pockets, and a total of 2 additional hydrogen bonds and 57 van der Waal interactions are made between inhibitor and protease. The μPlm/YO-2 structure reveals an unexpected binding mode ( Figure 1), in which the TXA moiety inserts into the S1 pocket and forms extensive canonical interactions with S736 and D735 at the base of the pocket in addition to the oxyanion hole (S741 and G739). Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here we report the crystal structures of plasmin in complex with the novel YO ( trans-4-aminomethylcyclohexanecarbonyl- l-tyrosine- n-octylamide) class of small molecule inhibitors. Thus, there is a need to develop specific inhibitors that target the protease active site. However, TXA lacks efficacy on the active form of plasmin. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. Accordingly, inhibitors of this system are clinically important. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis.