Formins are multidomain proteins that participate in a wide range of cytoskeletal processes that are required for cell polarity, cytokinesis, and morphogenesis in all eukaryotes. The defining feature of formin proteins is a highly conserved - 400 residue region, the Formin Homology 2 (FH2) domain, which has recently been found to directly nucleate actin filaments. Unlike the Arp2/3 complex, which nucleates branched filaments, formins induce unbranched filaments required for formation of diverse actin-containing structures including the contractile ring, actin cables, and stress fibers. The """"""""Diaphanous-related"""""""" formins (DRFs) are a subset of formins that are effectors for Rho-family GTPases. Because they reorganize the actin cytoskeleton in response to diverse cellular signals, formins are of central importance in cell biology and to human health. Defects in formin proteins result in failed cytokinesis and abnormal development. Defects in the human formin DFNA1 result in deafness. Our long-term goal is to understand at a structural level the regulated assembly of actin filaments by formin proteins. We seek to understand how the conserved formin FH2 domain nucleates and anchors actin filaments and to understand the intra- and inter- molecular interactions that regulate formin function. We are using X-ray crystallography and other biophysical methods to elucidate the structure and regulation of the yeast DRF Bni1 p. As described in the preliminary results, we have determined the crystal structure of the Bnilp FH2 domain. The structure of the FH2 domain reveals a novel """"""""tethered-dimer"""""""" architecture, in which the two halves of the dimer appear to be stably, but flexibly tied together. Based on our preliminary results, we hypothesize that the unusual tethered-dimer construction of the FH2 domain allows formins to nucleate actin filaments by stabilizing two actin subunits in the helical orientation found at the barbed end of actin filaments, and to stair-step on the barbed end of the nascent filament as it grows. Here we propose to test hypotheses generated by the structure through in vitro and in vivo structure/function studies, and to determine the structure of the FH2 domain in complex with actin in order to elucidate in detail the mechanism of actin assembly by formins. Furthermore, we will extend this work with crystallographic analysis of autoinhibitory regions within Bni1p in order to understand how they regulate the FH2 domain. ? ?
