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Decreased conformational stability in the oncogenic N92I mutant of Ras-related C3 botulinum toxin substrate 1

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Science Advances  07 Aug 2019:
Vol. 5, no. 8, eaax1595
DOI: 10.1126/sciadv.aax1595
  • Fig. 1 Mant-GDP dissociation rates of the wild type and the N92I mutant Rac1.

    (A) Mant-GDP dissociation rates of the wild type (left) and the N92I mutant (right). The data depicted as EDTA were measured in the presence of 1 mM EDTA. (B) Apparent Kd of Mg2+ of the wild type (left) and the N92I mutant (right). Kd values were calculated by using the equation, koff,GDP([Mg2+]) = A × Kd/([Mg2+] + Kd) + B, where A and B are constants. Each point reflects means ± SE of three independent experiments. The data for the wild type were originally reported in (7).

  • Fig. 2 Chemical shift differences between the wild type and the N92I mutant Rac1.

    (A) Plot of the normalized chemical shift differences between the wild type and the N92I mutant, Δδ. Δδ is calculated by the equation, Δδ = {(Δδ1H)2 + (Δδ15N /5)2}0.5. The residues with Δδ values larger than the SD (0.15 ppm) are colored red. (B) Overlay of the 1H-15N TROSY spectra of the wild type (black) and the N92I mutant (red), measured at 11.7 T (1H frequency, 500 MHz) and 25°C. The signals with Δδ values larger than 0.4 ppm are labeled. (C) Mapping of the residues with Δδ values larger than 0.15 ppm on the structure of Rac1 (PDB ID: 1DS6) (8). The residues with no data are colored gray.

  • Fig. 3 Conformational stability of Rac1.

    (A) Thermal denaturation curves of the wild type (black), the P29S mutant (green), and the N92I mutant (red), based on circular dichroism measurements observing the molar ellipticity at 222 nm. (B) Plots of the unfolding rates of the wild type (black) and the N92I mutant (red), measured in the presence of various concentrations of guanidine hydrochloride. (C) Schematic representations of the free energy landscape of the GDP binding reaction (left) and the folding reaction (right). The free energy landscapes of the wild type and the N92I mutant are colored black and red, respectively. The A and B states are the previously identified substates in the GDP-bound state, which differ in the affinity for Mg2+ (7).

  • Fig. 4 Experimental identification of the key hydrogen bond interaction that determines the conformational stability.

    (A) Plots of the amide and Trp ε-NH 1H temperature coefficients, Δδ/ΔT, of the wild type (top) and the N92I mutant (middle). The plot of the differences in Δδ/ΔT between the wild type and the N92I mutant is also shown (bottom). ppb, parts per billion. (B) Close-up view of the interactions formed between the α3 helix and the switch 2 region (PDB ID: 1DS6) (8). (C) Mant-GDP dissociation rates of the mutants of the residues that participate in the interaction formed between the α3 helix and the switch 2 region. (D) Mant-GDP dissociation rates of the Asn92 mutants. (E) Correlation plots of the mant-GDP dissociation rates of the Asn92 mutants versus the side-chain volumes (40).

  • Fig. 5 Molecular dynamics simulations of Rac1.

    (A) Close-up view of the wild-type Rac1 structure obtained after the 50-ns simulation (left). Plots of the distances between Asn92Hδ-Asp11Oδ1, Trp97Hε-Asp11Oδ1, and Trp97Hε-Asp11Oδ2 (center). Histogram of the distribution of Asp11 side-chain χ1 angles (right). (B) Close-up view of the N92I mutant Rac1 structure obtained after the 50-ns simulation (left). Plots of the distances between Trp97Hε-Asp11Oδ1 and Trp97Hε-Asp11Oδ2 (center). Histogram of the distribution of Asp11 side-chain χ1 angles (right).

  • Fig. 6 Schematic illustration of the decreased conformational stability in the N92I mutant and its activation mechanism.

    In the wild type (black), a hydrogen bond interaction (blue line) is formed between Asp11 and Trp97. In the N92I mutant (red), the side chain of Ile92 sterically blocks the hydrogen bond interaction (magenta circle). The loss of this hydrogen bond in the N92I mutant increases the GDP-bound ground-state energy and hence accelerates the GDP dissociation by lowering the activation energy along the GDP dissociation reaction coordinate.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax1595/DC1

    Table S1. MANT-GDP dissociation rates of the wild type and the N92I mutant in the presence of various concentrations of Mg2+.

    Table S2. MANT-GDP dissociation rates of the mutants of the residues from the α3 helix and the switch 2 region.

    Table S3. MANT-GDP dissociation rates of the mutants of Asn92.

    Fig. S1. Comparisons of the structures of Rac between the RhoGDI-bound state and the uncomplexed state.

    Fig. S2. Microsecond-order conformational exchange processes in the wild type and the N92I mutant.

    Fig. S3. Secondary structure populations of the wild type and the N92I mutant.

    Fig. S4. Structural characterizations of the P-loop.

    Fig. S5. Mant-GDP dissociation rates in the presence of guanidine hydrochloride.

    Fig. S6. Temperature-dependent changes in the 1H-15N TROSY spectra.

    Fig. S7. Characterizations of the structure and conformational stability of the W97A mutant.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. MANT-GDP dissociation rates of the wild type and the N92I mutant in the presence of various concentrations of Mg2+.
    • Table S2. MANT-GDP dissociation rates of the mutants of the residues from the α3 helix and the switch 2 region.
    • Table S3. MANT-GDP dissociation rates of the mutants of Asn92.
    • Fig. S1. Comparisons of the structures of Rac between the RhoGDI-bound state and the uncomplexed state.
    • Fig. S2. Microsecond-order conformational exchange processes in the wild type and the N92I mutant.
    • Fig. S3. Secondary structure populations of the wild type and the N92I mutant.
    • Fig. S4. Structural characterizations of the P-loop.
    • Fig. S5. Mant-GDP dissociation rates in the presence of guanidine hydrochloride.
    • Fig. S6. Temperature-dependent changes in the 1H-15N TROSY spectra.
    • Fig. S7. Characterizations of the structure and conformational stability of the W97A mutant.

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