Fig. 5. Conformation and dynamics of CHMP4 paralogs in solution.

(A) Hypothetical models of CHMP4 (2, 4, 32, 33), originally described by Vietri et al. (2). MIM, MIT-interacting motif, which binds to the MIT domain containing proteins (e.g., VPS4) (72); MIR, membrane insertion region. Helix α5 (pink) is likely responsible for CHMP4 autoinhibition. (B) Far-UV CD spectra (five scans), mean (solid line) and SD (shaded region), and (C) sedimentation profiles of $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$ (blue) and $\mathrm{CHMP}4{\mathrm{B}}_{121-224}^{\mathrm{S}184\mathrm{C}}$ (red). Arrow points to the dip at ~222 nm in the CD spectrum of $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$. NMR analysis of (D) $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$ and (E) $\mathrm{CHMP}4{\mathrm{B}}_{121-224}^{\mathrm{S}184\mathrm{C}}$, including TALOS-N–derived helical propensities (top), a comparison between experimental ³J_HN-Hα couplings against random coil values (middle), and ¹⁵N-R₂ profiles measured at 30°C (table S1). Unlike $\mathrm{CHMP}4{\mathrm{B}}_{121-224}^{\mathrm{S}184\mathrm{C}}$, $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$ showed greater helical propensity for residues 163 to 179, highlighted by semitransparent magenta rectangle. The corresponding drop in ³J_HN-Hα couplings and elevated ¹⁵N-R₂ values of this region confirmed the presence of a stable helix. The insert region of $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$ (see Fig. 3B) exhibited deviations from random coil ³J_HN-Hα couplings and elevated ¹⁵N-R₂ values (highlighted by semitransparent magenta rectangles), indicating a residual helical structure. Residues of the regions showing elevated TALOS-N–derived helical propensities (ranging between 0.25 and 0.56), indicating transient helical structures, are labeled. (F) Evidence of transient long-range interactions in CHMP4C using experimental PRE profiles of $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{G}154\mathrm{C}}$ (top) and $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{M}165\mathrm{C}}$ (bottom). The locations of paramagnetic label, MTSL, are marked with dashed lines. (G) Schematic of $\mathrm{CHMP}4{\mathrm{C}}_{121-233}^{\mathrm{S}191\mathrm{C}}$ (blue) and $\mathrm{CHMP}4{\mathrm{B}}_{121-224}^{\mathrm{S}184\mathrm{C}}$ (red) based on NMR results. Transient and stable helices are depicted as gray and magenta cylinders, respectively; numbers denote the corresponding residues. The CHMP4 helices based on the proposed models of full-length proteins [see (A)] are in parentheses.