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. 2018 Sep 15:148:9-18.
doi: 10.1016/j.ymeth.2018.04.005. Epub 2018 Apr 12.

An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution

John D Persons  1 , Shahid N Khan  1 , Rieko Ishima  2
Affiliations

An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution

John D Persons et al. Methods. .

Abstract

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, ΔCSPs, 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, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. 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.

Keywords: Deuteration; HSQC; NMR; NOE; Protein; Relaxation; Water.

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Figures

Fig. 1
Fig. 1
(A) Chemical structures of the HIV-1 PR inhibitor DRV and its analogue U10 (adopted from reference [11]), (B) comparison of DRV-PR (black) and U10-PR (red) 1H-15N NMR spectra [18], (C) graphical presentation of differences in 1H-15N combined chemical shift between DRV-PR and U10-PR (red, > 0.05 ppm; orange, 0.03 ppm) [18], and (D) conserved crystallographic water molecule positions at and around the PR-inhibitor interaction site. Protein graphics in (C) and (D) were generated using U10-bound PR crystal structure (PDB: 3O9I, [11]). In (C), residues numbers at the N- and C-termini and in an α-helix region are shown for Subunit A. In (D), residues at the subsequent region of the active site (residues 27 to 31), R87 (known to interact with D29), K8 (known to interact with two water molecules at the active site region), and the U10 inhibitor are shown as stick representation. Note, alphabet after the residue number, A or B, indicates the subunit A or B. Spheres (red, orange, pink, and yellow) indicate water molecule positions conserved in U2-, U3-, U7-, and U10-bound PR crystal structures (PDB: 3O9A, 3O9B, 3O9F, and 3O9I, respectively, [11]). Residue numbers near the water molecules are shown, and a water cluster near the inhibitor is highlighted with a dashed circle, without residue number indicated.
Fig. 2
Fig. 2
(A) 15N transverse relaxation rates, R2, of PR-DRV (circles) and PR-U10 (triangles), (B) its correlation, (C) het-NOE of PR-DRV (circles) and PR-U10 (triangles), and (D) its correlation. In (A and C), the data points in subunit A and subunit B are shown as black and open symbols, respectively. For comparison purpose, resonances that were not identified as either subunit were tentatively assigned to one of the two subunits.
Fig. 3
Fig. 3
15N half-filtered NOESY strips for the residues at the active site region of U10- and DRV-bound PR in (A) subunit A and (B) subunit B. Blue-circles indicate the water-NH NOEs. Red and green dashed horizontal lines indicate NOEs between PR and inhibitor, respectively.
Fig. 4
Fig. 4
Graphical presentation of (A) the active-site region of PR, showing the sites that have inhibitor-PR NOE connectivity (green dashed line) and intra-PR NOE connectivity (red dashed line) observed in Fig. 3, (B) a position of the conserved crystallographic water near T31A (orange dashed line), (C) its relative location in the PR-inhibitor complex, (D) the flap and the surrounding regions, showing the sites that have inhibitor-PR NOE (green dashed line), and the (E) its relative location in the PR-inhibitor complex. In (A-C), the graphics were generated using PR-U10 complex (PDB: 3O9I, [11]). In (A), stick shows residues of the active site region (residues 25-30). In (D and E), the graphics were generated using PR-U10 and PR-DRV (PDB: 3O9I and 1T3R, [11,54]). Other notations are the same as those in Fig. 1D.
Fig. 5
Fig. 5
15N half-filtered NOESY strips at the flap region and residue 82 for both subunits A and B. Symbols are notations are the same as those described in Figure 3.
Fig. 6
Fig. 6
(A) Overlay of a 2D water-amide NOE spectrum (black) and a water region of the 1H-15N slice of 3D 15N-half filtered NOESY experiment (red), and (B) a 2D water-amide ROE spectrum. In (A), residue numbers of the peaks that are likely from labile side chains are indicated by small font. In (B), black and red show positive (ROE) and negative (exchange) peaks, respectively.
Fig. 7
Fig. 7
Histograms of filter/unfilter ratio in the wNOE spectra for residues (A) in the β-sheet and (B) in other regions at different T2 filter times, and (C) a residue profile of the filter/unfilter ratio calculated using Eq. (1), for 26 ms T2 filter data. In (A and B), the T2 filter times were varied: open bars, 14 ms; black bars, 20 ms; gray bars, 26 ms. In (C), different symbols were used to express the data points of backbone amides: in the β-sheet, circle: in the flap region, rectangle; those that undergoes exchange, triangle; others, cross. Aspartic acid and threonine residues, many of which interact with the inhibitors, have been removed in (A) and (B) while included in (C).
Fig. 8
Fig. 8
(A) Comparison of filter/unfilter ratios (A) for residues in PR-U10 versus those in PR-DRV, in which P1′ and P2′ moiety differ, (B) for residues in PR-U10 versus those in PR-U7, in which only P1′ moiety shares, and (C) for residues in PR-U2 versus those in PR-U7, in which only the P2′ moiety is shared [11]. All filter/unfilter ratios here were calculated using Eq. (1) for 20 ms T2 filter data. In each panel, open circles and filled triangles represent residues in subunits A and B, respectively. Residues at/within the active site loop (residues 25-32), flap region (residues 46-56), and P1 loop (77-84) are shown by black, red, and blue, respectively.
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