Dynamic Consequences of Inactivating the Catalytic Serine 195 to Methionine in the Human Urokinase-Type Plasminogen Activator (uPA)

Abstract

Human urokinase-type plasminogen activator (uPA) is a serine protease that has implications in various health processes including anticoagulation, angiogenesis and cancer. The catalytic site of uPA is made by a histidine (H57), an aspartic acid (D102) and a serine (S195), that last one being responsible for the nucleophilic attack that starts the peptide bond hydrolysis. Studying the dynamics of uPA is key to develop strategies to control the activity of this serine protease, however, analyzing this protein by many biophysical techniques is very challenging because it self-degrades under the experimental conditions, such as high protein concentration or long incubation times. We hypothesized that mutating S195 to methionine would yield a stable catalytically inactive mutant that would biophysically behave similarly to the wildtype. Site-directed mutagenesis was used to create the mutant. The mutant protein was expressed in E. coli as inclusion bodies, refolded in vitro and purified by a series of chromatography steps including Ni-affinity chromatography, aprotinin-affinity chromatography and size exclusion chromatography. The dynamics in solution of the S195M mutant and the wildtype were studied by hydrogen-deuterium exchange mass spectrometry (HDXMS). There were no significant differences in most of the protein, except in the 140s loop. In this loop, there are three peptides that have less protection in the S195M mutant versus the wild type protease. This is significant due to the fact that the 140s loop is at the entrance to the active site of the protease. These results suggest that the S195M mutant is causing allostery in the 140s loop.