An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution
Publication date: Available online 12 April 2018
Source:Methods
Author(s): John D. Persons, Shahid N. Khan, Rieko Ishima
This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (
PR), we previously evaluated amide chemical shift differences, ?CSPs, of
PR bound to darunavir (DRV) compared to
PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor’s chemical moiety variation [Khan, S. N., Persons, J. D, Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652-1662]. However, ?CSPs are not ideal for investigating subtle
PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ?CSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar
PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15N-half filtered NOESY spectra of
PR bound to two highly analogous inhibitors and assessed NOEs between
PR amide protons and inhibitor protons, between
PR amide protons and hydroxyl side chains, and between
PR amide protons and water protons. We also verified the
PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes.
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An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution
Linear Mode
