A Structural and Biochemical Study of the Interaction Between Actin and the Mammalian Formin FRL2

Formins are a class of proteins that influence the rate of actin filament nucleation and elongation. Mammals possess 15 formin isoforms, providing a myriad of possibilities for regulating actin-based structures in cells. The dimeric formin homology 2 (FH2) domain is capable of altering the nucleation rate of new actin filaments and subsequently influences filament elongation via direct interaction with the barbed end of an actin filament. The FH2 domain moves processively with the barbed end as the filament elongates. Our goal in this study is to examine FMNL3's interaction with actin to uncover mechanistic details of formin regulated actin elongation. The FH2 domain of FMNL3 that forms a stable interaction with tetramethylrhodamine-malamide labeled actin (TMR-actin) and has been crystallized and the 3.3 A resolution structure has been solved. The crystal structure indicates that FMNL3 dimerizes to form a ring around the barbed end of two actin subunits. The formin/actin interface is comprised of multiple interactions that are found in three distinct regions of the FH2 domain. Mutational analysis identified key residues in FMNL3/actin binding as well as those essential for elongation activity. The study of these residues has led us to propose a more detailed model for the interaction of the FH2 domain with actin. A previous study with Bni1p, revealed an FH2 dimer conformation spanning three actin subunits. This structure supports the model of a formin tracking with the growing end of an actin filament by taking steps with the addition of each monomer. The FMNL3/actin complex represents an intermediate step in the formin stepping mechanism in which two actin subunits are aligned side-by-side surrounded by a symmetric FH2 dimer. This structure reveals mechanistic details of FH2 mediated actin filament elongation via processive capping.