Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core

Abstract

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ∼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ∼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Keywords: Actin assembly; Autotransporter; FH2; Formin; Spotted fever group Rickettsia.

Figure 1.. Sample preparation and overall structure of Sca2.

(A) The domain organization of Rickettsia conorii Sca2 (Uniprot Q92JF7). The expressed Sca2 construct (residues 34-1515) used in this study is indicated. SS: signal sequence, NRD: N-terminal repeat domain, PRD: Proline-rich domain, CRD: C-terminal repeat domain, AC: Auto chaperone, TD: Translocator domain. (B) SDS-PAGE of Sca2 elutions following Ni-NTA and StrepTrap affinity purification. (C) Glycerol gradient chromatogram and SDS-PAGE analysis of the boxed fractions. (D) Representative negative stain TEM micrograph of Sca2. (E) Representative cryo-EM micrograph of Sca2. (F) Cryo-EM density map of Sca2 shown from three views, and the domains of Sca2 rigid-body fit into the density.

Figure 2.. Sca2 comparison with formins.

Sca2 from our cryo-EM analysis compared with crystal structures of various formins. (A) Top- Sca2 model, with NRD residues 34-400 in red, residues 401-670 in yellow, and CRD residues 1090-1355 shown in green. Bottom- Sca2 model showing only residues 34-670, with CRD residues hidden. (B-E) Crystal structures of different formins. FH2 monomers are colored red or yellow for a straightforward comparison, and in (B-C) actin is colored in blue (B) Top- FMNL3 bound to actin (PDB 4EAH [37]). Bottom- Same as top, actins hidden. (C) Top- Bni1p bound to actin (PDB 1Y64 [35]). Bottom- Same as top, actins hidden. (D) DAAM1 (PDB 2Z6E [36]). (E) Bni1p (PDB 1UX5 [34]).

Figure 3.. Proposed models for actin assembly by Sca2, Formins, and ParMRC.

(A) Sca2 nucleates actin by encircling two (or more) actin monomers, and elongation could proceed by an insertional mechanism, using its PRDs and profilin-actin. (B) Formins nucleate actin by encircling two (or more) actin monomers, and elongate by the addition of actin subunits from profilin-actin via their PRDs in their flexible FH1 domains. (C) ParR binds both the barbed end of the actin-like ParM (via it’s C-terminus) and ParC (via it’s N-terminus). The free ParM binding sites on unoccupied ParR are involved in bringing in ParM monomers to the barbed end for processive elongation. In the elongation model, ParRC is shown as a single semi-circular shape for simplicity. See text for details.