NMR analyses of ALIX-CHMP4 interactions and CHMP4 conformtation and dynamics

Figure 4A: 1H/15N chemical shift perturbation profiles of 100 μM 15N/2H-labeled Bro1 on addition of 300 μM unlabeled CHMP4 constructs; CHMP4C(121–233, S191C) and CHMP4B(121–224,S184C). <br> Figure 4C: 1H/15N and 13C’/15N chemical shift perturbation profiles of 150 μM 15N/13C-labeled PRD(703–800)Strep in the presence of 450 μM unlabeled CHMP4 constructs. <br> Figure 5F: Evidence of transient long-range interactions in CHMP4C paralog using experimental PRE profiles (shown as paramagnetic-labeled vs. diamagnetic-labeled 1H/15N cross-peak height ratios) of 15N-labeled CHMP4C(121–233,G154C) and CHMP4C(121–233,M165C). <br> Figure 6A and 6B: The reduction in 1H/15N cross-peak heights of 150 μM 15N-labeled (A) CHMP4C(121–233,S191C) and (B) CHMP4B(121–224,S184C) as a function of increasing amount of unlabeled Bro1.<br> <br> <br> Figure 6C and 6D: The perturbations in 1H/15N chemical shifts of 150 μM 15N-labeled (C) CHMP4C(121–233,S191C) and (D) CHMP4B(121–224,S184C) as a function of increasing amount of unlabeled PRD(703–800)Strep.<br> <br> <br> Figure 6I, 6J, and 6K: The reduction in 1H/15N cross-peak heights of 30 μM 15N/2H-labeled (I) (CHMP4C121–233,S191C), (J) CHMP4B(121–224,S184C), and (K) CHMP4C(121–224,S184C, I225A, L228A, W231A) in the presence of 90 μM unlabeled ALIX(1–868,P801G)Strep.

图4A：100 μM的15N、2H双同位素标记Bro1蛋白，在加入300 μM未标记CHMP4构建体（CHMP4C(121–233, S191C)与CHMP4B(121–224, S184C)）后的1H/15N化学位移扰动图谱。
图4C：150 μM的15N、13C双同位素标记的Strep标签融合PRD(703–800)蛋白，在450 μM未标记CHMP4构建体存在下的1H/15N与13C羰基/15N化学位移扰动图谱。
图5F：通过15N同位素标记的CHMP4C(121–233,G154C)与CHMP4C(121–233,M165C)的顺磁弛豫增强（Paramagnetic Relaxation Enhancement, PRE）图谱（以顺磁标记与抗磁标记的1H/15N交叉峰高度比呈现），证实了CHMP4C旁系同源蛋白中存在瞬时长程相互作用。
图6A与6B：150 μM的15N同位素标记的（A）CHMP4C(121–233,S191C)与（B）CHMP4B(121–224,S184C)的1H/15N交叉峰高度，随未标记Bro1蛋白添加量递增的变化情况。

图6C与6D：150 μM的15N同位素标记的（C）CHMP4C(121–233,S191C)与（D）CHMP4B(121–224,S184C)的1H/15N化学位移扰动值，随未标记PRD(703–800)Strep蛋白添加量递增的变化情况。

图6I、6J与6K：30 μM的15N、2H双同位素标记的（I）CHMP4C(121–233,S191C)、（J）CHMP4B(121–224,S184C)以及（K）CHMP4C(121–224,S184C, I225A, L228A, W231A)，在90 μM未标记Strep标签融合ALIX(1–868,P801G)蛋白存在下的1H/15N交叉峰高度降低情况。