Research ArticleSTRUCTURAL BIOLOGY

Conformational landscape alternations promote oncogenic activities of Ras-related C3 botulinum toxin substrate 1 as revealed by NMR

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Science Advances  13 Mar 2019:
Vol. 5, no. 3, eaav8945
DOI: 10.1126/sciadv.aav8945
  • Fig. 1 GDP dissociation rates and NMR spectra of the wild-type Rac1 and the P29S mutant.

    (A) Structure of the Mg2+-binding site. Crystal structure of the GDP-bound Rac2 in the Rac2-Rho GDI complex [Protein Data Bank (PDB) ID: 1DS6] (8). The switch regions are colored cyan, and Pro29 is colored red. The close-up view of the Mg2+-binding site is shown on the right. (B) Mant-GDP dissociation rates of the wild-type Rac1 (left) and the P29S mutant (right) in the presence of various concentrations of Mg2+. The data depicted as EDTA were measured in the presence of 1 mM EDTA. (C) Apparent Kd of Mg2+ for the wild type (left) and the P29S mutant (right). Each point reflects mean ± SE of three independent experiments. (D) Overlay of the 1H-13C HMQC spectra of the wild type (black) and the P29S mutant (red), measured at 14.1 T (600-MHz 1H frequency) and 25°C. In the NMR experiments, the R66E background mutation is introduced to suppress the self-association. ppm, parts per million. (E) Mapping of the methyl groups with marked chemical shift differences onto the structure of Rac1. The normalized chemical shift differences, Δδ, are calculated by the equation, Δδ = {(Δδ1H)2 + (Δδ13C/5.6)2}0.5. The methyl groups with Δδ larger than 0.05 ppm are colored red.

  • Fig. 2 GDP dissociation in the T35A mutant and conformational exchange processes of Rac1.

    (A) Mant-GDP dissociation rates of the T35A mutant in the presence of various concentrations of Mg2+. The data depicted as EDTA were obtained in the presence of 1 mM EDTA. (B) Apparent Kd of Mg2+ for the T35A mutant. (C) Overlay of the 1H-13C HMQC spectra of the wild type (black), the P29S mutant (red), and the T35A mutant (blue), measured at 14.1 T (600-MHz 1H frequency) and 25°C. The methyl groups highlighted in the spectra are mapped onto the structure of Rac1. (D) CPMG RD results of the Ala42β methyl groups of the wild type, the P29S mutant, and the T35A mutant. Close-up views of the 1H-13C HMQC spectra, the chemical shifts of states A and B, and the exchange parameters are also shown. (E) Linear correlation plot of the apparent Kd of Mg2+ versus the population of the B state. In the NMR experiments, the R66E background mutation is introduced to suppress the self-association.

  • Fig. 3 Mant-GDP dissociation rate measurements and NMR analyses in the presence of Rho-GDI.

    (A) Mant-GDP dissociation rates in the presence of various concentrations of Rho-GDI. Mg2+ was added to the final concentration of 0.1 mM. We confirmed that the purified Rho-GDI inhibits GDP dissociation from Rac1. (B) Overlay of the 1H-13C HMQC spectra in the absence (black) and presence (purple) of Rho-GDI, measured at 14.1 T (600-MHz 1H frequency) and 25°C. One-dimensional cross sections of the Val168γ1 signal are shown. The direct interaction was verified from the NMR spectral changes of Rac1 upon the addition of stoichiometric amounts of Rho-GDI. (C) Close-up views of the 1H-13C HMQC spectra in the absence (black) and presence (purple) of 200 μM Rho-GDI. Cross marks denote the chemical shifts of states A and B, calculated from the CPMG RD and HSQC/HMQC analyses in the absence of Rho-GDI. (D) Overlays of the 1H-13C HMQC spectra of the wild type (WT) in the presence of 200 μM Rho-GDI (purple), the T35A mutant (blue), the wild type in the absence of Rho-GDI (black), and the P29S mutant (red). In the NMR experiments of the wild type in the absence of GDI, the T35A mutant, and the P29S mutant, the R66E background mutation is introduced to suppress the self-association. (E) 13C SQ CPMG RD profiles in the absence (black) and presence (purple) of 200 μM Rho-GDI. CPMG RD experiments were performed at 14.1 T (600-MHz 1H frequency) and 25°C.

  • Fig. 4 Intramolecular PRE experiments.

    (A) Plots of intramolecular PREs observed from the MTSL-conjugated E31C. Bar graphs show the experimentally observed PREs, green lines show the back-calculated PREs using the crystal structure of the Rho-GDI complex, and magenta lines show the back-calculated PREs calculated from two-state ensemble structural calculations. Asterisk denotes a Trp side-chain ε-NH signal. Small differences between the observed PREs and the back-calculated PREs were observed in the region around Val51, which is located adjacent to the flexible region (residues 24 to 47) defined in the ensemble calculations. We assume that these differences are attributed to the fact that the side-chain structures are slightly changed from the crystal structure in the B state because these residues are located in the border between the movable and fixed residues. Long-range PREs observed on Val44NH and Met45ε methyl protons are illustrated on the structure. In the PRE experiments, the R66E background mutation is introduced to suppress the self-association. (B) Linear correlation plots of the PREs observed on Ala13β methyl, Val44NH, and Met45ε methyl protons versus the population of the B state estimated under the experimental conditions. Error bars were calculated on the basis of the signal-to-noise ratios of the spectra. (C) Structural comparison between the structure of the A state that corresponds to the crystal structure of the Rho-GDI complex and the structure of the B state obtained from the two-state ensemble structural calculations under the PRE-based distance restraints. The structure of the B state represents the lowest-energy structure, and a main-chain atomic probability density map of 20 lowest-energy structures at the contour level of 0.1 is shown as a transparent surface model. (D) Close-up views of the switch1 structures of states A and B. The Cα atoms of Pro29 are colored red.

  • Fig. 5 1H-15N RDC experiments.

    Bar graphs represent the experimentally observed RDCs, and orange lines represent the calculated RDCs using the crystal structure of the Rho-GDI complex. The residues with no data are shown with gray backgrounds. In the experiment, the R66E background mutation is introduced to suppress the self-association. The measurements were performed at 14.1 T (600-MHz 1H frequency) and 20°C.

  • Fig. 6 Conformational landscapes of the wild-type Rac1 and the P29S mutant.

    The structure of the A state represents the crystal structure of the Rho-GDI complex, and the structure of the B state represents the lowest-energy structure obtained from ensemble structural calculations under the PRE-based distance restraints. The exchange parameters obtained from CPMG RD and HSQC/HMQC analyses are shown.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav8945/DC1

    Table S1. MANT-GDP dissociation rates of Rac1.

    Fig. S1. 1H-13C HMQC spectra of the wild-type Rac1 and the T35A mutant.

    Fig. S2. 1H-15N TROSY spectra of the wild-type Rac1 and the P29S mutant.

    Fig. S3. CPMG RD analyses of the wild-type Rac1.

    Fig. S4. Summary of the global fitted results of the CPMG RD and HSQC/HMQC analyses.

    Fig. S5. Summary of the intramolecular PRE experiments.

    Fig. S6. Summary of the global fitted results of the CPMG RD and HSQC/HMQC analyses of the Cys-less mutant of Rac1.

    Fig. S7. Comparison between the structures of states A and B obtained from the two-state ensemble structural calculations.

    Fig. S8. MD simulations of Rac1.

    Fig. S9. Mg2+ affinity and NMR spectra of the R66E mutant of Rac1.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. MANT-GDP dissociation rates of Rac1.
    • Fig. S1. 1H-13C HMQC spectra of the wild-type Rac1 and the T35A mutant.
    • Fig. S2. 1H-15N TROSY spectra of the wild-type Rac1 and the P29S mutant.
    • Fig. S3. CPMG RD analyses of the wild-type Rac1.
    • Fig. S4. Summary of the global fitted results of the CPMG RD and HSQC/HMQC analyses.
    • Fig. S5. Summary of the intramolecular PRE experiments.
    • Fig. S6. Summary of the global fitted results of the CPMG RD and HSQC/HMQC analyses of the Cys-less mutant of Rac1.
    • Fig. S7. Comparison between the structures of states A and B obtained from the two-state ensemble structural calculations.
    • Fig. S8. MD simulations of Rac1.
    • Fig. S9. Mg2+ affinity and NMR spectra of the R66E mutant of Rac1.

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