title: Structure and activity of full-length formin mDia1.
abstract: Formins are a conserved family of actin assembly-promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on formins has been limited to fragments. This has left open key questions about the structure, activity and regulation of intact formin proteins. Here, we isolated full-length mouse mDia1 (mDia1-FL) and found that it forms tightly autoinhibited dimers that can only be partially activated by RhoA. We solved the structure of autoinhibited mDia1-FL using electron microscopy and single particle analysis. Docking of crystal structures into the three dimensional reconstruction revealed that the fork-shaped N-terminal diaphanous inhibitory domain-coiled coil domain region hangs over the ring-shaped formin homology (FH)2 domain, suggesting that autoinhibition results from steric obstruction of actin binding. Deletion of the C-terminal diaphanous autoregulatory domain extended mDia1 structure and activated it for actin assembly. Using total internal reflection fluorescence microscopy, we observed that RhoA-activated mDia1-FL persistently accelerated filament elongation in the presence of profilin similar to mDia1 FH1-FH2 fragment. These observations validate the known activities of FH1-FH2 fragments as reflecting those of the intact molecule. Our results further suggest that mDia1-FL does not readily snap back into the autoinhibited conformation and dissociate from growing filament ends, and thus additional factors may be required to displace formins and restrict filament length.
authors.affiliation: Biology Department, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
authors.forename: Sankar
authors.initials: S
authors.lastname: Maiti
authors.forename: Alphee
authors.initials: A
authors.lastname: Michelot
authors.forename: Christopher
authors.initials: C
authors.lastname: Gould
authors.forename: Laurent
authors.initials: L
authors.lastname: Blanchoin
authors.forename: Olga
authors.initials: O
authors.lastname: Sokolova
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Goode
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation; D000080865:Formins; D051379:Mice; D008840:Microfilament Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D002352:Carrier Proteins; C491464:Diap1 protein, mouse; D000080865:Formins; D008840:Microfilament Proteins; D020742:rhoA GTP-Binding Protein