Structural Basis of CYRI-B Direct Competition with Scar/WAVE Complex for Rac1

Abstract

Highlights d CYRI-B structure reveals homology to CYFIP of the Scar/ WAVE complex d CYRI-B shares a conserved interface with CYFIP for Rac1 interaction d CYRI proteins form autoinhibited dimers that compete for Rac1 binding d Rac1-CYFIP model shows potential steric clashes of RAC1 with CYFIP

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

“…We next queried whether CYRI-A could interact with active RAC1. Using the I-TASSER protein prediction tool (Zhang, 2008;Roy et al, 2010;Yang and Zhang, 2015) along with the recently solved crystal structures of CYRI-B (Yelland et al, 2021;Kaplan et al, 2020), CYRI-A was predicted to contain an amphipathic N-terminal α-helix connected via a flexible linker to a bundle of 12 α-helices forming the domain of unknown function (or DUF1394 domain; Fig. 2 A).…”

CYRI-A regulates macropinocytic cup maturation and mediates integrin uptake, limiting invasive migration

“…CYRI-A, encoded by the FAM49A gene, has previously been linked to non-syndromic oral clefts and craniofacial abnormalities (Azevedo et al, 2020;Chen et al, 2018;Leslie et al, 2016), suggesting an important role in developmental and perhaps morphogenetic processes, but its cellular and molecular function has not been studied. Here, we model CYRI-A into the recently solved structure of CYRI-B (Kaplan et al, 2020;Yelland et al, 2020) and we conclude that CYRI-A likely adopts a similar structure to CYRI-B but may also have important differences. We show biochemically that CYRI-A interacts with active RAC1 using similar amino acid residues, but with a higher affinity than CYRI-B.…”

“…We next queried whether CYRI-A could interact with active RAC1. Using the I-TASSER protein prediction tool (Roy et al, 2010; Yang et al, 2015; Zhang, 2008) along with the recently solved crystal structures of CYRI-B (Yelland et al, 2020, Kaplan et al, 2020), CYRI-A is predicted to contain an amphipathic N-terminal α-helix connected via a flexible linker to a bundle of 12 α-helices forming the Domain of Unknown Function (or DUF1394 domain) (Fig. 2 A ).…”

“…2B and (Fort et al, 2018)) and the two arginine residues at position 159/160 (for CYRI-A) or 160/161 (for CYRI-B) that are essential for active RAC1 binding to CYRI-B (Red box, Fig. 2B ) and (Fort et al, 2018; Yelland et al, 2020). Using an in vitro pulldown assay where GST-tagged RAC1 was immobilised onto beads, we showed that the region of CYRI-A that corresponds to the “RAC1 Binding Domain” or RBD of CYRI-B (Fort et al, 2018) (amino acid 29-319) robustly and consistently interact with active Q61L RAC1 (Fig.…”

“…It is important to emphasize that all of these events happen locally on the macropinocytic cups as they are forming with a high degree of spatiotemporal control. We queried whether CYRI-A's ability to bind active RAC1 is important for its recruitment to nascent macropinosomes by using a RAC1-binding defective mutant (Yelland et al, 2020) with arginine 159 and 160 mutated to aspartic acid (P16-mCh-CYRI-A-RRDD). We co-transfected COS-7, HEK293T or CHL-1 cells with the wild-type P16-GFP-CYRI-A and either the wild-type or the mutant RRDD construct of P16-mCherry-CYRI-A ( Fig.…”