Abstract
Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.
Highlights
Fragment-based drug discovery (FBDD) has proven to be an effective method to develop medicinal drugs (Erlanson 2012)
We demonstrated that ligands that are in fast exchange between bound and free state can exhibit transferred pseudocontact shifts (PCS) caused by lanthanoid tags on the protein, which can be used to determine a low-resolution model of the ligand in the binding site, provided that a structure of the protein is available and under the assumption that ligand binding does not result in backbone conformational changes
Ntd-HSP90 was tagged at three sites using the Caged Lanthanoid NMR probe #5 (CLaNP-5), containing either Lu3+ as a diamagnetic control or Y b3+ as a paramagnetic center
Summary
Fragment-based drug discovery (FBDD) has proven to be an effective method to develop medicinal drugs (Erlanson 2012). FBDD is based on finding very small molecules that bind to the target with a large average binding energy per heavy atom (~ 0.3 kcal/mol/heavy atom). Such fragments still have a low affinity and need to be elaborated to molecules with more negative binding free energies, either by linking fragment hits or by growing them (Bohacek et al 1996; Hajduk and Greer 2007). Journal of Biomolecular NMR (2018) 71:275–285 diffraction of crystals or NMR spectroscopy The former technique is most commonly used but NMR is an alternative for structure determination and offers complementary information. Less demanding methods are based on transferred NOEs, paramagnetic relaxation enhancements (PRE) or pseudocontact shifts (PCS) to obtain information about the ligand bound state while benefitting from the narrow linewidths of the ligand in the free state (Viegas et al 2011; Guan et al 2013; Jahnke et al 2000, 2001)
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