Date Published: February 8, 2018
Publisher: Public Library of Science
Author(s): Tobias Kromann-Hansen, Eva Louise Lange, Ida K. Lund, Gunilla Høyer-Hansen, Peter A. Andreasen, Elizabeth A. Komives, Eugene A. Permyakov.
The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a pivotal role in fibrinolysis in the extracellular space as initiator of a protein cascade eventually leading to generation of another trypsin-serine protease, plasmin. Plasmin, in turn, acts directly to degrade fibrin and indirectly, and relatively nonspecifically, to activate matrix-metalloproteases which then degrade collagen . Under normal physiological conditions, the proteolytic activity of uPA is involved in processes such as wound healing. However, under abnormal pathophysiological conditions, the activity of uPA is implicated in tissue remodeling in several diseases including rheumatoid arthritis, progression of cancer and atherosclerosis [2–6].
HDXMS analyses were performed with the catalytic serine protease domain of muPA (muPA16-243) in the following states: ligand free muPA16-243 (apo-muPA16-243), EGR-cmk-bound muPA16-243, mU1-bound muPA16-243 and mU3-bound muPA16-243. We have recently reported the HDXMS uptake plots for selected peptides in the muPA16-243 and EGR-cmk bound muPA16-243 variants . Here, we perform the HDXMS experiments again in order to provide the complete peptide coverage map and corresponding uptake plots of all pepsin-generated peptides in muPA16-243 and EGR-cmk bound muPA16-243. The proteins or complexes were diluted into buffered D2O and the resulting mass increase due to incorporation of deuterium was monitored as a function of time (0, 0.5, 1, 2, 5 min). Deuterium incorporation was localized to various regions of muPA16-243 by mass analysis of peptides produced by pepsin proteolysis, resulting in the identification of 36 overlapping peptides that together cover 92% of the muPA16-243 sequence (Fig 1A).
When compared to apo- muPA16-243, EGR-cmk-bound muPA16-243 showed decreased amide exchange in regions corresponding to the N-terminal activation loop, the 140s, the 180s, and the 220s loops. The four loops are interconnected through three patches of polar interaction networks (patch I, II and III) (Fig 6). In muPA16-243, patch I interconnects the N-terminal base of the 140s loop with the 180s loop (Fig 6). Residues Lys192 and Asp194, which is adjacent to the oxyanion hole residues (Gly193 and Ser195), are in direct contact with the residues Gly142 and Lys143 of the 140s loop. Patch II interconnects the C-terminal base of the 140s loop to the N-terminal activation loop. Residues Leu155, Lys156 and Met157 of the 140s loop are in direct contact with residues Ile16, Gly18, Glu20 and Thr22 in the N-terminal sequence (Fig 6). Patch III interconnects the 140s loop and the 220s loop. Glu146 contacts Glu222, which is in vicinity of residues of the S1 specificity pocket (Gly218) (Fig 6). Our HDXMS results clearly show that the N-terminal sequence, the 140s, the 180s, and the 220s loops is highly dynamic in apo- muPA16-243. Binding of EGR-cmk, however, dampens exchange throughout the loops presumably by strengthening the interconnectivity of stabilizing interactions in patches I, II and III. Importantly, our HDXMS data revealed a relatively high amide exchange level (71%) in the peptide covering the N-terminal Ile16 suggesting that Ile-16 may be mostly solvent-exposed. This observation is in good agreement with the notion that apo- muPA16-243 crystallizes in a conformation with a solvent-exposed Ile16 .