K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins

Figures

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  Figure 1. Properties of purified K336I monomeric actin. (A) The release rate of ε-ATP from K336I and WT actin monomers was monitored by measuring the decrease in fluorescence intensity after the addition of regular ATP (1 mM) to 20 µM K336I or WT G-actin that had been incubated with ε-ATP. The dissociation rates of ε-ATP from K336I mutant actin and WT actin were 0.009 ± 0.001 s−1 and 0.025 ± 0.005 s−1, respectively (mean ± SE, N = 3). There is a statistically significant difference between K336I and WT actin (t-test, p < 0.01). (B) K336I and WT actin (4 µM) were treated with 0.5 µg/ml subtilisin for the indicated time, and analysed by SDS-PAGE. Arrows indicate intact actin bands (42 kDa) and arrowheads indicate digested actin bands (36 kDa). MW, molecular weight markers. Full-length gel image is shown in Supplementary Fig. S3. (C) Quantitation of intact actin bands from Coomassie-stained gels, including the one shown in (B). After 3 minutes of subtilisin digestion, 52.1 ± 4.3% of K336I G-actin was intact, vs. 29.6 ± 6.4% of WT G-actin (mean ± SE, N = 3).

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  Figure 2. Co-sedimentation of K336I actin filaments and alpha-actinin ABD. Solid lines show data fit using the following equation: [alpha-actinin ABD]bound/[F-actin] = [alpha-actinin ABD]free/Kd + [alpha-actinin ABD]free. The dissociation constants (Kd) of alpha-actinin ABD from actin filaments were as follows: K336I homo-filaments (blue circles), 30.0 ± 2.7 µM; WT homo-filaments (red squares), 15.1 ± 2.0 µM; and K336I/WT co-filaments (black triangles), 25.2 ± 2.2 µM (mean ± SE, N = 4). There is a statistically significant difference between WT homo-filaments and K336I homo-filaments (t-test, p < 0.001) as well as between WT homo-filaments and K336I/WT co-filaments (t-test, p < 0.01).

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  Figure 3. Sliding velocities of K336I and WT actin filaments on myosin II-HMM and myosin V-HMM. Velocities (mean ± SE) on surfaces of myosin II-HMM were as follows: K336I homo-filaments, 1.00 ± 0.02 µm/s (N = 137); WT homo-filaments, 1.90 ± 0.03 µm/s (N = 110); and K336I/WT co-filaments, 1.55 ± 0.03 µm/s (N = 165). Velocities on surfaces of myosin V-HMM were as follows: K336I homo-filaments, 0.204 ± 0.003 µm/s (N = 40); WT homo-filaments, 0.212 ± 0.004 µm/s (N = 40); and K336I/WT co-filaments, 0.202 ± 0.002 µm/s (N = 40). Asterisks indicate a statistically significant difference compared to WT homo-filaments (t-test, p < 0.001). Statistically insignificant differences are indicated by “n.s.”.

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  Figure 4. Cofilin binding. (A) Co-sedimentation of 2.5 µM cofilin with 5 µM K336I homo-filaments, 5 µM WT homo-filaments, and K336I/WT co-filaments consisting of 5 µM WT actin and 5 (1:1), 1.7 (0.33:1), or 0.5 µM (0.1:1) K336I mutant actin was analysed by SDS-PAGE. sup, supernatant; ppt, pellet after ultracentrifugation. Asterisks indicate contaminating proteins present in the BSA solution. Full-length gel image is shown in Supplementary Fig. S8. (B) Densitometric analysis of three Coomassie-stained SDS-gels, including the one shown in (A), determined that 5.6 ± 2.6%, 52.2 ± 12.4%, 69.5 ± 13.6%, and 93.9 ± 6.5% (mean ± SE) of cofilin co-sedimented with K336I homo-filaments, 1:1, 0.33:1, and 0.1:1 co-filaments, respectively. The amount of cofilin in each pellet was normalized to the amount in the pellet of WT homo-filaments.

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  Figure 5. Copolymerisation of cofilin–actin fusion protein with K336I actin affects cleavage of the D-loop in the fusion protein. As shown in our previous report31, the fusion protein polymerises and depolymerises normally in a salt-dependent manner, and the cofilin moiety in the fusion protein has a pH-sensitive depolymerisation and severing activities of actin filaments. These functional properties of the cofilin moiety are similar to that of normal cofilin. Thus, there is seems to be no steric problem between the actin and cofilin moieties in the fusion protein in terms of interaction with other actin molecules. (A) Schematic drawing showing the detection of structural changes of the actin moiety in the fusion protein when copolymerised with K336I actin. In a co-filament, when K336I actin protomers affect the structure of the WT actin moiety of the fusion protein, they reduce the affinity of that actin moiety for cofilin moiety, causing its D-loop to have reduced susceptibility to subtilisin cleavage. The D-loop of actin (except for the actin moiety in the fusion protein) is not shown. (B) Subtilisin digestions of K336I/cofilin–actin co-filaments or WT/cofilin–actin co-filaments.…