Structural studies of proteins by NMR spectroscopy and modelling : characterization of the dengue virus protease using pseudocontact shifts

Abstract

In this thesis, NMR spectroscopy is used to characterise the conformation of the dengue virus protease in complex with a small molecular weight inhibitor. The dengue virus protease, as many other interesting drug targets, displays a complex NMR spectrum with highly overlapped and poorly resolved cross-peaks in the 15N-HSQC spectrum. New methodologies that rely on selective isotope labelling and the introduction of several paramagnetic centers at different positions in the protein yielded NMR assignments of the dengue virus protease, permitting subsequent structural analysis. The strategies presented in this thesis open an avenue for the efficient analysis of NMR spectra with only partially resolved peaks. In combination with an independently determined 3D structure of the target protein, the information contained in the resolved cross-peaks of the 15N-HSQC spectrum was sufficient to assess the fold of the dengue virus protease. The dengue virus protease is a serine protease consisting of the NS2B cofactor and the NS3 protease domain (NS3pro). Depending on the presence of an inhibitor, it can assume two very different conformations. In the most frequently used expression construct, both components are connected by an artificial eight-residue peptide linker. Crystal structures of the protease without an inhibitor revealed an open conformation of the protease, with the NS2B cofactor located far from the active site. At the start of this thesis, no high-resolution structure of the dengue virus protease was available in complex with an inhibitor, but NMR data suggested the occurrence of a large structural change upon inhibitor binding. Two different paramagnetic tags were introduced at three different positions of the protein. In combination with two different paramagnetic lanthanide ions per tag, twelve datasets of pseudocontact shifts were obtained. The resulting structural restraints confirmed the accuracy of a homology model built of the dengue virus protease in complex with an inhibitor. Compound binding to DENpro as well as the reaction rates of the related West Nile virus protease (WNVpro) were studied by established fluorescence assays with WNVpro experiencing an increase in the reaction rate after incorporation of an unnatural amino acid. The structure of the dengue virus protease without covalent link between NS2B and NS3pro in solution was assessed by the NMR methods previously established in this thesis. Unexpectedly, in this new construct, the protease was found to assume the closed conformation even in the absence of an inhibitor. This suggests that the open conformation is an artefact introduced by the peptide linker. Rational drug design approaches should therefore target the closed conformation rather than the open state of the dengue virus protease. In addition, this thesis describes the structure calculation of a small protein, a RRM1 domain, using the CS-Rosetta protocol which was performed as a side project. A recent upgrade of the Rosetta software package includes PCS data as structure restraints in the structure calculation. Together with the strategies for selective resonance assignment of resolved cross-peaks developed in this thesis, this sets the groundwork for an automated interpretation of PCSs observed in protein NMR spectra in the future. Show more