Mechanism of actin thin filament pointed-end elongation by leiomodin

Figures

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  Fig. 1. Cryo-EM structures of Lmod2 at the PE. a Domain diagrams of human Lmod2 and Tmod1: TMBS tropomyosin-binding site, ABS1 actin-binding site-1, DBS D-loop-binding site, LRR leucine-rich repeat domain, PRD proline-rich domain, WH2 WASP-homology 2 domain. Note that only the N-terminal portion of Tmod1’s ABS1 is conserved in Lmod2. b Cryo-EM map of F-actin (mid-filament structure) at ~2.0-Å resolution, including close-ups of the nucleotide-binding site showing the bound ADP, Mg²⁺ ion (green), and water molecules (red), as well as representative regions of the map. c Perpendicular views of the one-Lmod2 at the pointed end (PE) map: LRR, yellow; actin subunits, gray, except P and P-1, which are colored in different shades of green. d Close-up (red circle in c) of Lmod2’s DBS and map, showing its interaction with P-1’s D-loop. e The DBS-stabilized conformation of P-1’s D-loop (light green) compared to that of a mid-filament subunit (gray). f P is rotated as a whole by ~6° away from the filament axis compared to a mid-filament subunit (gray). g P (left) exhibits a G-actin conformation (outer domain rotated ~20°, red arrow, relative to an F-actin subunit, gray), while P-1 (right) adopts a…

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  Fig. 2. Binding of two Lmod2 molecules at the PE and subunit recruitment. a, b Perpendicular views of the one-Lmod2+actin and two-Lmod2+actin maps (colored as in Fig. 1, with the extra actin in magenta). c Close-up (blue circle in b) of the first Lmod2 DBS and map, showing interactions with P + 1’s H-cleft and P-1’s D-loop. d Close-up of the first LRR in the one-Lmod2+actin structure, showing interactions with four actin subunits. e P + 1 adopts a near G-actin (pink) conformation, distinct from F-actin (gray), in both the one-Lmod2+actin and two-Lmod2+actin structures. f Close-up of the second LRR in the two-Lmod2+actin structure. g P + 1 is rotated ~6.5° away from the filament axis in the two-Lmod2+actin structure, but not in the one-Lmod2+actin structure. h P adopts an intermediate conformation between G-actin (pink) and F-actin (gray) in the two-Lmod2+actin structure (but not in the one-Lmod2+actin structure), with the outer domain rotated ~11° relative to F-actin.

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  Fig. 3. Model of Lmod2-mediated elongation. At the PE, we observe four distinct structural states: free PE, one-Lmod2, one-Lmod2+actin, and two-Lmod2+actin, interpreted as consecutive steps in the elongation mechanism. First, Lmod2’s LRR binds at the PE, interacting with P, P-1, and P-2, with the binding energy and thermal fluctuations at the PE allowing it to stabilize high-energy states, including a G- to F-actin transition in P-1 and an overall ~6° rotation of P away from the filament axis. An actin subunit (P + 1) is then recruited through interactions with P, P-1, and the DBS of the LRR. A second Lmod2 binds on the opposite side of the filament, opening another actin recruitment site at the PE of P; P-1 adopts an intermediate conformation between G- and F-actin, and P + 1 rotates ~6.5° away from the filament axis. Although not captured in the structures, we propose that as P fully transitions toward an F-actin conformation, the first LRR is sterically displaced (as shown in Fig. 1g) and dissociates, allowing P-1’s D-loop to insert into the H-cleft of P + 1. This initiates a new elongation cycle, returning to the one-Lmod2 state, with P as the new P-1 and P + 1 as the new P. The…