19F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2

Abstract

The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*–E equilibrium and providing no evidence that free thrombin is zymogen-like.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR

“…The structural alignment of the protein backbone of 7M8Q or 7M8R with that of the sMMOH:MMOB complex (PDBID: 6VK5) yields root-mean-square deviation (RMSD) values of 0.097 and 0.149 Å, respectively, indicating that the structures are nearly identical. The overall structure of the sMMOH:MMOB complex and the conformational changes induced by MMOB have been discussed in detail elsewhere, and thus they are not described further here. , It is important to note that the conservation of structure upon substitution of 5FW for tryptophan has been commonly observed in other proteins. ,− …”

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy

“…F-Trp residues deliver readily accessible site-specific information from simple 1D 19 F NMR spectra. This has been exploited with great success in a multitude of applications such as measuring diffusion coefficients of proteins in live cells, , protein folding/unfolding equilibria, − and dynamics arising from ligand binding, at protein interfaces, or as reflected in interconversion rates between different conformations . Furthermore, the 19 F chemical shifts of F-Trp residues are highly sensitive to their chemical environment, which is advantageous for ligand binding studies. − 19 F NMR signals can still be detected by solution NMR of systems up to 500 kDa .…”

Assessing the applicability of 19F labeled tryptophan residues to quantify protein dynamics

“…Numerous impressive examples are available in the literature where 19 F NMR methods have been exploited to extract information regarding protein motions (Kitevski-LeBlanc and Prosser 2012 ). These include studies of a fold-switch in the mycobacterial glycosyltransferase PimA (Liebau et al 2020 ), calmodulin (Hoang and Prosser 2014 ), the nonstructural protein 1 of influenza A virus (Aramini et al 2014 ), GPCRs (Liu et al 2012 ; Kim et al 2013 ; Didenko et al 2013 ; Manglik et al 2015 ; Frei et al 2020 ; Pan et al 2022 ), the periplasmic glucose/galactose receptor (Luck et al 1996 ), diacyl-glycerol kinase (Shi et al 2011 ), a cold shock protein (Overbeck et al 2020 ), alkaline phosphatase (Hull and Sykes 1974 ), the chaperone PapD (Bann et al 2002 ), spider silk (Sarker et al 2016 ), the protease trypsin (Ruben et al 2020 ) and the Xrn2 exoribonuclease (Overbeck et al 2022 ). However, to our knowledge, it has never been systematically evaluated whether the incorporation of 19 F labels has an effect on the native protein dynamics, despite reports that show that 19 F labeling can have an effect on protein structure and stability (Xiao et al 1998 ; Minks et al 1999 ; Acchione et al 2012 ; Dalvit and Vulpetti 2016 ).…”