Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly

Abstract

In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell-cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function.

Keywords: CYRI-B; FAM49B; SAD; actin assembly; cell-motility regulator; crystal structure.

Figure 1

Crystal structure of CYRI-B. Cartoon representation of native CYRI-B from R. typus showing the N-terminal (green), medial (yellow) and C-­terminal (blue) subdomains. The myristoylation site of the protein (Fort et al., 2018 ▸) is indicated. A schematic linear organization of the protein subdomains is represented underneath.

Figure 2

Oligomeric state of CYRI-B. (a) Native PAGE (left) and SDS–PAGE (right) gels of native (Nat.) and SeMet-derivatized (Se) CYRI-B proteins. Molecular masses of protein standards (lane M) are shown in kDa. (b) Size-exclusion chromatography profiles of the indicated proteins.

Figure 3

Comparison of human and whale shark CYRI-B reveals high sequence similarity. (a) Alignment of whale shark and human protein sequences, where divergent amino acids are displayed in red and identical residues are represented by dots. The secondary structure of whale shark CYRI-B is shown above the sequence using the same colour code as in Fig. 1 ▸. The alignment was generated with the NCBI BLAST program (Altschul et al., 1997, 2005 ▸). (b) Side chains of residues that are dissimilar between the whale shark and human proteins are represented as spheres on each subdomain of the whale shark CYRI-B structure. Non-conservative differences between the two proteins are shown in bold in (a) and (b). (c) Views of the electrostatic surface of whale shark (left) and human (Phyre homology model; right) CYRI-B proteins. A 360° rotation tour is presented in Supplementary Movie S3.

Figure 4

Comparison of whale shark CYRI-A and CYRI-B. (a) Radical substitutions between CYRI-A and CYRI-B as indicated by Clustal Omega (Sievers et al., 2011 ▸) are shown as blue spheres on the CYRI-B structure. The corresponding residues in CYRI-B are indicated. (b) Electrostatic surfaces of CYRI-A (Phyre homology model) and CYRI-B (crystal structure). (c) Sequence alignment of multiple CYRI-A and CYRI-B proteins highlighting residues 73 and 109 (R. typus CYRI-B numbering). Abbreviations are as follows: If, Ictalurus furcatus; Rt, R. typus; Xl, Xenopus laevis; Ch, Crotalus horridus; Hs, H. sapiens; Fc, Felis catus; Ms, Mus musculus. The full sequence alignment is provided in Supplementary Fig. S3(a).

Figure 5

CYRI-B and CYFIP1 present a high degree of structural similarity. (a) Side-by-side view of the CYRI-B (left) and CYFIP1 DUF1394 domain (PDB entry 3p8c; right) structures. The colour code of CYRI-B is as in Fig. 1 ▸. The presence of an extra hairpin in CYFIP1 is indicated. (b) Secondary-structure alignment of the N-terminal (green), medial (yellow) and C-terminal (blue) subdomains of the two proteins. The r.m.s.d. value and number of C^α atoms are shown beside each alignment (from SUPERPOSE; Krissinel & Henrick, 2004 ▸). (c) Top, overall structure of the WAVE regulatory complex (WRC; PDB entry 3p8c). The DUF1394 domain of CYFIP1 is coloured as in (a). Bottom left, close-up view of the 304–314 hairpin of CYFIP1 showing residues involved in hydrogen-bond interactions with the rest of the protein (grey). Bottom right, superposition of CYRI-B on the DUF1394 domain of CYFIP1 showing the absence of the 304–314 hairpin. An asterisk indicates a steric clash.

Figure 6

Predicted association of Rac1 with CYRI-B and CYFIP1. (a) Site-directed mutagenesis data mapped onto the R. typus CYRI-B crystal structure. Mutations in CYRI-B (black labels) that impair (red spheres) or do not affect (blue spheres) its function in vivo (Shang et al., 2018 ▸) are shown. Green labels indicate mutations in the CYFIP1 DUF1394 domain that perturb the association of the WRC complex with Rac1 (Chen et al., 2010 ▸). (b) Close-up views of the predicted Rac1-interacting regions of CYRI-B (left) and CYFIP1 (right), coloured as in Fig. 5 ▸. Residues leading to effective mutations in (a) are represented as cyan sticks with the corresponding mutated residue indicated. Electron densities of CYRI-B side chains are shown as a blue mesh contoured at a σ level of 1.