These authors contributed equally to this work and are joint first authors
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These authors contributed equally to this work and are joint senior and corresponding authors
Version 1
: Received: 14 August 2023 / Approved: 15 August 2023 / Online: 15 August 2023 (05:12:23 CEST)
Version 2
: Received: 23 August 2023 / Approved: 23 August 2023 / Online: 23 August 2023 (09:57:06 CEST)
Quek, A.J.; Cowieson, N.P.; Caradoc-Davies, T.T.; Conroy, P.J.; Whisstock, J.C.; Law, R.H.P. A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen. Int. J. Mol. Sci.2023, 24, 14258.
Quek, A.J.; Cowieson, N.P.; Caradoc-Davies, T.T.; Conroy, P.J.; Whisstock, J.C.; Law, R.H.P. A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen. Int. J. Mol. Sci. 2023, 24, 14258.
Quek, A.J.; Cowieson, N.P.; Caradoc-Davies, T.T.; Conroy, P.J.; Whisstock, J.C.; Law, R.H.P. A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen. Int. J. Mol. Sci.2023, 24, 14258.
Quek, A.J.; Cowieson, N.P.; Caradoc-Davies, T.T.; Conroy, P.J.; Whisstock, J.C.; Law, R.H.P. A High-Throughput Small-Angle X-ray Scattering Assay to Determine the Conformational Change of Plasminogen. Int. J. Mol. Sci. 2023, 24, 14258.
Abstract
Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant 'closed' conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an 'open' form which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high throughput (HTP) 96-well SAXS assay setup. This method enables us to determine the Kopen value, which is used to compare ligands’ effect on Plg conformation directly. Based on our analysis using Plg GII, we have found that the Kopen for ε-aminocaproic acid (EACA) is approximately three times greater than that of Tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin and receptor Plg‑RKT. Our findings suggest that, besides the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Commenter: Ruby Law
Commenter's Conflict of Interests: Author