Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium

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Science Advances  30 Sep 2020:
Vol. 6, no. 40, eabd0480
DOI: 10.1126/sciadv.abd0480
  • Fig. 1 Structures of Bcl-xL in unligated (top) and ABT-737–bound (bottom) states.

    The structure of bound ABT-737 is shown in the magenta stick representation. Cryptic site on Bcl-xL is indicated by a red shadow. Apo structure, 1maz; ABT-737 complex, 2yxj.

  • Fig. 2 NMR analysis of the interaction between Bcl-xL and ABT-737.

    (A) Heteronuclear multiple quantum correlation (HMQC) spectrum of 100 μM [Ile-δ1, Met-ε, Leu-δ2, Val-γ2 methyl-1H13C] Bcl-xL in unligated (black) and excess amount of ABT-737 (120 μM, red). (B) Normalized CSPs for each residue upon binding to ABT-737. (C) Mapping of substantially perturbed residues (Δδ > 0.2 ppm, red; Δδ > 0.1 ppm, orange) on the ribbon representation of Bcl-xL in ABT-737–bound conformation. ABT-737 is shown in a magenta stick representation.

  • Fig. 3 Presence of the open cryptic site conformation in unligated Bcl-xL.

    (A) Temperature-dependent population changes in the Ile χ2, Leu χ2, Met χ3, and Val χ1 rotameric states between 288 and 308 K in apo Bcl-xL. The populations in the rotameric states were calculated from the equations shown in Materials and Methods. (B) Mapping of residues that showed substantial temperature dependence of their rotameric states. The methyl moieties with >2 SDs of rotameric population shift in response to the temperature variation are highlighted in red. (C) Van’t Hoff plot of three resonances that showed the largest temperature-dependent 13C chemical shift changes. (D) Methyl CPMG RD experiments of unligated Bcl-xL. The residues that showed substantial Rex (>5 Hz) contributions to the transverse relaxation of the 13C methyl moiety are mapped on the structure of Bcl-xL. For those residues in the cryptic site (Leu108, Ile114, and Leu150), the RD plot and their global fitting are shown.

  • Fig. 4 Structural characterization of open cryptic site mutant F143W.

    (A) Overlaid spectra of the unligated F143W mutant (cyan) and the unligated (black) and ABT-737–bound (red) WT Bcl-xL. Residues with substantial allosteric CSPs upon ABT-737 binding are shown. The peak positions of the residues that were not subjected to the analysis are indicated by *, **, #, and ## for Leu17, Leu90, Val10, and Leu162, respectively. Unligated and ABT-737–bound peaks are connected by a dashed line. (B) Methyl RD experiments with the unligated F143W mutant. The residues with substantial Rex (>5 Hz) contributions to the 13C methyl R2 were mapped on the structure of Bcl-xL, along with the mutation site. For the residues in the core of the cryptic site (Ile114, Leu162, and Leu166), the RD plots and their global fittings are shown.

  • Fig. 5 Efficient screening of the cryptic site–bound peptide by the F143W mutant.

    (A) Comparison of the enrichment factors in each round of phage-display screening. The ratio of hit clone against the whole clone cfu was shown for each panning round. (B) Normalized CSP induced by the hit peptide; 160 μM peptide was added to 100 μM WT Bcl-xL. (C) Mapping of the residue that experienced normalized CSP > 0.01 ppm. (D) Correlation plot of the ABT-737–induced allosteric 13C CSPs against the peptide-induced 13C CSPs.

Supplementary Materials

  • Supplementary Materials

    Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium

    Yumiko Mizukoshi, Koh Takeuchi, Yuji Tokunaga, Hitomi Matsuo, Misaki Imai, Miwa Fujisaki, Hajime Kamoshida, Takeshi Takizawa, Hiroyuki Hanzawa, Ichio Shimada

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