Small-Molecule Binding Sites on Proteins Established by Paramagnetic NMR Spectroscopy
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- 作者：Jia-Ying Guan ; Peter H. J. Keizers ; Wei-Min Liu ; Frank L枚hr ; Simon P. Skinner ; Edwin A. Heeneman ; Harald Schwalbe ; Marcellus Ubbink ; Gregg Siegal
- 刊名：The Journal of the American Chemical Society
- 出版年：2013
- 出版时间：April 17, 2013
- 年：2013
- 卷：135
- 期：15
- 页码：5859-5868
- 全文大小：579K
- 年卷期：v.135,no.15(April 17, 2013)
- ISSN：1520-5126
文摘
Determining the three-dimensional structure of a small molecule鈥損rotein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D 1H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.