A structural model of the profilin–formin pacemaker system for actin filament elongation

Figures

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  Figure 1. Actin filaments docked onto formin:actin heterodimers support a ‘stepping second’ model. The two panels on the left depict the result of superimposing the terminal subunit of a 6-mer (in lemon and orange) of the helical actin filament (PDB code: 6DJO) onto actin molecule (N−1) bound to the trailing FH2(T) domain (light blue) in the Bni1:actin heterodimer (PDB code: I64Y). With this alignment, actin(N) bound to FH2(L) (in dark blue) does not fit the helical symmetry of the filament but is instead coplanar with actin(N−2). Actin subunits in the filament are not N-acetylated. Superposing β-actin from the crystal structure of profilin-β-actin (PDB code: 2BTF) onto actin(N) places profilin (in red) in a shallow surface pocket where it forms significant salt-bridges with FH2(L) and the N-terminus of actin(N). The coplanarity of actin(N) (in brown) with actin(N−2) is seen in the bottom panels (E,G) where FH2 has been removed. The two panels on the upper right show the terminal subunit of a 6-mer of the helical actin filament docked onto actin(N) of the mammalian formin FMNL3:actin crystal structure (PDP code: 4EAH). For consistency in relating the Bni1 and FMNL3 filament-bound…

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  Figure 2. Central role for profilin R88 in coordinating actin polymerization by FH2. The boxed region of panel (A) shows profilin residues R88 and H119 sequestering Y169, the keystone residue in the longitudinal actin(N)-actin(N−2) interface. Actin residues K373, C374 and F375 form part of the pocket surrounding Y169. The arrow points to the prominent profilin ‘wing’ R88-T97 (purple). Panel (B), boxed region, shows (left) the interaction between actin(N) residue D2 and the FH2(L) linker residues K1410 and R1411. Detailed view (right image of panel B) of profilin–actin interface shows the salt-bridge between R88 and profilin residue E116 and an electrostatic interaction involving actin residues Y169, K372 and profilin residue H119. The boxed region of panel (C) shows the sidechain of profilin R88, held in position by a salt-bridge with E167 of actin(N), lying along the base of the profilin ‘wing’ (purple: R88–T97) that contacts the turns at the ends of the actin-binding FH2 knob helices (A) and (B).

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  Figure 3. Profilin release associated with polymerization. Panel (A) shows actin(N)-actin(N−2) as bound by the Bni1 and FMNL3 FH2 dimers, (left) and (right), respectively. In Bni1, the actin molecules are coplanar, while in FMNL3 they are related by the helical symmetry of the cryo-EM structure (PDB: 6DJO). During the release of profilin from actin(N) in the Bni1 structure the D-loop in SD2 extends towards actin(N−2) as seen in the FMNL3 structure. Y169, the central residue in the longitudinal contact between actin(N) and actin(N−2), is exposed with the release of profilin. Panel (B) depicts the profilin-β-actin structure (PDB: 2BTF) showing the proximity of the profilin binding site to the hinge and shear region of actin. The FH2 knob helix A occupies part of this site with profilin (not shown). The right image illustrates the environment of Y169 in the longitudinal bond of the filament.

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  Figure 4. Transition State for the Formin Elongation Reaction. The Bni1:actin FH2 heterodimer with bound F-actin (left in Fig. 1) is shown with a profilin–β-actin dimer derived from the crystal structure (space group 19; PDB: 2BTF) docked onto the penultimate actin(N−1) subunit. The center panel in (A) illustrates how the binding of the dimer positions actin(N+1) at the entrance to the polymerase at the open end with the axial spacing (5.5 nm) of the actin helix. The PA and PC poly-l-proline tracts of FH1 have been docked onto profilins bound to actin(N+1) and actin(R) respectively. The path (faint blue, middle panel) of the FH2(T) linker (1400–1420) in the open space below the actin(N−1)–actin(R) interface is dictated by its interaction with the actin(N−1) N-terminus, positioning the lasso on the opposite side of the dimer from its knob domain. Panel (A) show in three orientations a possible path for the FH1 polypeptide as it emerges (in the carboxy to amino direction) from the top of the FH2(T) lasso. Panel (B) shows the transition-state in the same orientation as the central panel of (A) but clipped by a plane the cuts through the center of the structure removing from view actin(N−1)…

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  Figure 5. Profilin controls the timing of the elongation reaction. The front and back views of the two co-crystal structures afford views of the interaction between FH2 domains and actin at four steps (A–B, C–D, E–F, G–H) during the elongation reaction. Panels (A) and (B) show two orientations of the Bni1 FH2-actin heterodimer (modeled as in Fig. 1) stabilized by the binding of profilin–actin(N) to FH2(L). Actin(N) and actin(N−2) are coplanar. In panels (C) and (D) profilin has been released from actin(N) during the migration of FH2(T) towards the barbed end creating a binding pocket for actin(N+1). Actin(N) rotates 13° and docks into the helical niche formed by actin(N−1) and actin(N−2). In panels (E) and (F) the insertion of profilin–actin(N+1) results in the coplanar alignment of actin(N+1) with actin(N−1). In panels (G) and (H), following the release of profilin, actin(N+1) rotates and docks onto the barbed end of the filament with the closing of a small gap (indicated by red dotted circle) between actin(N−1) and actin(N+1). Note that to model the four steps in the reaction sequence as viewed from the front, the colors of FH2(L) and FH2(T), as well as those of the filament strands,…

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  Figure 6. Schematic of the formin elongation reaction. Panel 1 represents the state of the actin polymerase after the insertion of actin(N) in a coplanar orientation with actin(N−2) on the same strand (orange). Its associated profilin (red) is held in place by its interaction with actin(N) and FH2(L). The actin(R)-actin(N+1) dimer has bound FH1 via the affinity of their profilin molecules for poly-l-proline tracts (PC and PA). Panel 2 depicts the binding of actin(R) to actin(N−1) which places actin(N+1) at the entrance to its binding pocket. Panel 3 shows the second stepping event where the dissociation of FH2(T) and its migration towards the barbed end ferries its linker-lasso (light blue) towards the post of FH2(L) as profilin is released from actin(N). The bond between actin(R) and actin(N−1) is broken as actin(N+1) is drawn into the pocket formed by the knob of FH2(T) and the post of FH2(L). Panel 4 represents the state where actin(N) has rotated and docked into the helical niche formed by actin(N−1) and actin(N−2). Actin(N−1) and actin(N+1) are coplanar, actin(R) has been carried off by FH1 and the FH2(T) lasso has bound to the FH2(L) post. Panel 5 illustrates step 2 in the…