1. Probing contacts of inhibitor locked in transition states in the catalytic triad of DENV2 type serine protease and its mutants by 1H, 19F and 15 N NMR spectroscopy
Peter Agback, Esmeralda Woestenenk, Tatiana Agback BMC Mol Cell Biol. 2020 May 25;21(1):38. doi: 10.1186/s12860-020-00283-0.
Background: Detailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides. Results: Monitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other. Conclusions: Our reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using 19F NMR spectroscopy.
2. The Structure of the Zika Virus Protease, NS2B/NS3pro
Rolf Hilgenfeld, Jian Lei, Linlin Zhang Adv Exp Med Biol. 2018;1062:131-145. doi: 10.1007/978-981-10-8727-1_10.
In this chapter, we first briefly review the history of Zika virus (ZIKV) over the past 70 years since its discovery. We then focus on the ZIKV NS2B/NS3 protease, a major potential target for anti-ZIKV therapeutics. We describe the structure of the complex between Zika virus NS2B-NS3 protease and a peptide boronic-acid inhibitor that we determined in early 2016. We then review other structural studies on the Zika virus protease, which have been published in the past few months. Three different types of construct for the protease have been investigated by X-ray crystallography and NMR spectroscopy: the traditional "linked" construct comprising the NS2B cofactor, a Gly4SerGly4 linker, and the NS3pro chain; a construct where the linker has been replaced by Lys-Thr-Gly-Lys-Arg, which leads to autocleavage; and the bimolecular "unlinked" protease consisting of the NS2B cofactor segment and NS3pro. In complex with an inhibitor, the protease adopts a closed, "active" conformation with the NS2B chain wrapped around the NS3pro and contributing to the S2 pocket. In the ligand-free state, the Gly4SerGly4-linked enzyme adopts an open or relaxed conformation, with the C-terminal half of the NS2B cofactor highly flexible and disordered. Very surprisingly, however, the "unlinked", bimolecular protease has been reported to adopt the closed conformation in the crystal, even though, apparently, no peptide was bound to the substrate-binding site. The Gly4SerGly4-linked enzyme has been used successfully in drug discovery efforts.
3. (19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions
Naima G Sharaf, Angela M Gronenborn Methods Enzymol. 2015;565:67-95. doi: 10.1016/bs.mie.2015.05.014. Epub 2015 Jun 16.
(19)F solution NMR is a powerful and versatile tool to study protein structure and protein-ligand interactions due to the favorable NMR characteristics of the (19)F atom, its absence in naturally occurring biomolecules, and small size. Protocols to introduce (19)F atoms into both proteins and their ligands are readily available and offer the ability to conduct protein-observe (using (19)F-labeled proteins) or ligand-observe (using (19)F-containing ligands) NMR experiments. This chapter provides two protocols for the (19)F-labeling of proteins, using an Escherichia coli expression system: (i) amino acid type-specific incorporation of (19)F-modified amino acids and (ii) site-specific incorporation of (19)F-modified amino acids using recombinantly expressed orthogonal amber tRNA/tRNA synthetase pairs. In addition, we discuss several applications, involving (19)F-modified proteins and (19)F-containing ligands.
