Formin Mouse Diaphanous Homolog 1 Structural Analysis
created by Philip Dinh
Formin Mouse Diaphanous Homolog 1 (pdb ID = 3OBV, mDia1) from Mus musculus, is a protein involved in the regulation of actin and the microtubule cytoskeleton. As a protein in the formin family, which are known as conserved actin regulators, MDia1 specializes in polymerizing actin and regulating actin filaments through an activity known as processive capping. MDia1 acts upon actin filaments to accelerate nucleation, and interacts with the terminal ends of these long filaments to modulate length and orientation of their growth. Through its functioning in the formation of stress fibers, actin cables, and other elements of the cytoskeletal system, mDia1 exhibits significant roles in cytokinesis, cytoskeleton maintenance, and cell migration. The molecular weight of the mDia1 tetramer is 557,372 Da, and its isoelectric point (pI) is 5.43, as provided by the ExPASy Database (ExPASy, 1).
The structure of mDia1 correlates directly to its function and interactions with its specific ligands. Physiologically, mDia1 equilibrates between a homodimer and a homotetramer in solution, consisting of either two of four identical subunits. In the commonly formed asymmetric unit the tetrameric configuration is preferred, interacting as a complex of two mDia1 dimers. This arrangement consists of four chains of the protein monomer, which has a primary structure consisting of 1255 amino acids with a combined molecular weight of 139,343 Da per unit (Uniprot, 1).
Each of these subunits is broken down into five distinct domains: Formin Homologies 1-3(FH1 –FH3), coiled coil(CC), and the diaphanous autoinhibitory domain(DAD). The three FH domains, which are characteristically present in most of the formin class proteins, can be further subdivided into smaller regions and are highly conserved in the FH1 and FH2 domains, though the FH3 domain shows a greater degree of variation across related proteins. FH3 covers residues 73 – 451, and consists of the Rho GTPase binding domain (G) from residues 73 – 130, the diaphanous inhibitory domain (DID) from residues 131-377, and dimerization domain(DD) from residues 378-451. As an effector of the Rho family of GTPases, the G domain is involved in the binding of GTP-bound Rho proteins, inducing a conformational change on the characteristic DID-DAD complex of mDia1. The dimerization domain is responsible for the association and consolidation of the protein subunits to form a dimer, and for subsequently stabilizing the subunit-subunit interactions to maintain the dimer and potential tetramer. The adjacent CC region further stabilizes the dimer, and exhibits an asymmetric conformation in full length mDia1. Following the CC region, FH1 covers residues 570-753, and is responsible for mediating interactions with a wide variety of proteins, most significantly Profilin. The FH1 domain, through this Profilin binding, is involved in the acceleration of processive capping and thus rapid growth of actin filaments. The FH2 domain is composed of a Lasso region covering residues 754-805, a Linker region covering residues 806-834, and the Core region from residues 835-1163. This domain forms a ring-like FH2-DAD dimer at the C-terminus where two actin bridge elements are connected through the linker regions. Each actin bridge element(BE) is capable of holding two actin monomers, resulting in the nucleation of new filaments and processive capping in this region of the protein. The DAD region extending from the bridge element can complex with DIDs of the same or different subunits within the tetramer, in the latter case forming the autoinhibited arrangement necessary to actin regulation (Otomo, 12896).
The secondary structure of mDia1 is composed primarily of alpha helices(red), with a few beta sheets(yellow) and several 3/10 helices(purple) interspersed. The DID-DD region consists of 80.7% a-helices and 1.5% 3/10 helices. The DID assumes its folded architecture with five repetitions of its three helix element into a superhelix. The DD region, also highly helical, consists of three helices which associate with their counterparts on another subunit to create a dimer of six helices. Intertwining of this helical bundle in conjugation with the stabilization from the CC region forms the (DID-DD)2 dimer, and when DID is in its free form (pdb ID = 2BNX) separates the two DID axes at a 64o angle (Lammers, 4176). The FH2 domain consists of 69.2% a-helices and 5.5% 3/10 helices. There exists only one beta sheet at either of the DID-DD and FH2 domains, composed of only 1% of the residues in their respective regions.
Formin mDia1’s primary function, actin regulation, is mediated by its autoinhibitory complex formed by the DID-DD and FH2-DAD regions of the protein. The C-terminal DAD binds the N-terminal DID, which in the tetrameric arrangement forces the actin-binding sites on each BE to face inward to the center of the structure. The tertiary structure of the dimer at its FH2-DAD complex reveals a knob and post site on the core of the FH2 homology. The linker region, extending from the knob, wraps around the post at its lasso section to form the ring conformation. Autoinhibition through DID-DAD binding results in an orientation that blocks the active site of the ring by leaving no space for actin monomers (pdb ID = 2BAP). Due to partial overlap between the Rho and DAD binding sites on DID, this autoinhibition is relieved when GTP-bound Rho proteins bind to the DID and adjacent G region, displacing the DAD and allowing the FH2-DAD ring dimer to appropriately initiate actin assembly (Otomo, 273).
This autoinhibited arrangement is formed first by the bridge element, which via the c-terminal alpha-helix(aT) is able to extend from the post domain by 10 turns, connecting to a loop at Leu-1169. This connects the BE to the DAD at a region of alpha helices from Val-1181 to Gly-1192, which is already bound to the DID until Phe-1195. The aT-DAD loop is a slightly flexible element which preserves the asymmetric tetramer in its autoinhibited state. In this conformation, the critical residues for actin-binding function are occupied by contacts with other interfaces on the protein. Ile-845, a critical knob site residue, interacts with the aT of another bridge element in a heavily Gln-based chain, at the residues Gln-1163, Gln-1164, Lys-1165, and Gln-1168. The positively charged residues on the lasso region and post site, Arg-769, Arg-764, and Arg-975 are neutralized by Glu-227, Glu-229, Glu-230, Glu-268, Glu-272, Glu-276, and Glu-279, the highly negative stretch on the long axis of the DID. Alternative tetrameric configurations also exist based on this premise of autoinhibition by binding of the FH2-DAD active site. Dissociation of (DID-DD)2-(DID-DD)2 interactions in interchanging between tetrameric configurations elucidates the dimer-tetramer equilibrium observed in solutions of natural mDia1 (Otomo, 12896).
Full length mDia1 in its dimeric configuration is a possibility arising from a similar process as in the tetrameric configuration, and may involve actin monomer binding. The most likely mechanism for dimeric inhibition invokes the flexibility of the aT loop which gives the structure more degrees of freedom, preventing complete occlusion of actin-binding sites. NMR spectra of chemical shifts in two exposed isoleucine sidechains of the FH2 homology revealed a broadening of peaks in Ile-1170 upon its binding to DID, suggesting an equivalent contact to the tetrameric configuration. Broadening of Ile-845 resonance upon introduction of tetramethylrhodamine-actin, in the presence of BE-DAD-DID binding via Ile-1170, indicated an interaction between the actin monomer and the knob site. Accessibility of actin-binding sites in the full-length mDia1 model suggests that dimeric autoinhibition may be carried out by means of a joint complex of an actin monomer bound to and inhibiting the BE-DAD-DID system (Otomo, 12896).
The two primary ligand components for mDia1 are Rho GTPases, such as RhoA or RhoC-GMPPNP, and Actin. Actin monomers are manipulated by the active site of mDia1 and utilized for nucleation, filament growth, and processive capping for cytoskeletal and microtubule development. Members of the Rho family of GTPases, RhoC exhibited as a chief example, regulate mDia1 through the use of its ‘switch’ regions, which allows it to displace the DID-DD-DAD complex (pdb ID = 1Z2C) by interacting with the G and DAD subdomains of FH3 (Rose,513). The RhoC complex separates the DID axes by 154o, whereas they are separated by 126o in the DID-DD-DAD structure. Sucrose was also present as a ligand, but was an experimental addition and residual side product of crystallization and x-ray diffraction.
Human Dishevelled-associated activator of morphogenesis-1, HDaam1 (pdb = 2J1D), has an approximate 29% sequence similarity with mDia1, and almost identical protein subdomains. The results of protein blast(E = 3E-48), a program that compared and cross-referenced proteins based on amino acid sequence homology, showed that HDaam1 has a similar primary structure to mDia1 (BLAST, 1). The DALI server, a database designed to finding proteins of similar tertiary structures of the query protein, did not reveal any comparison for HDaam1 for pdb = 2J1D, but did show tertiary similarity for related human formins of a different conformation (Dali, 1). Formin HDaam1 shares key sites with mDia1 common in actin-regulatory activity. HDaam1 also shares a tethered dimer structure and potential tetrameric subunit organization. Functionally, HDaam1 exhibits binding though a lasso region and bridge element, inhibiting the N and C-terminus with a BE-DAD-DID linkage almost identical to mDia1. Structural differences in HDaam1 and mDia1 are few but functionally important in the rate of actin assembly. In its dimeric configuration the actin binding surfaces of HDaam1 are completely occluded in an autoinhibited conformation, stabilized by two beta strands not present in mDia1. This weakens the actin-related activity of HDaam1 in comparison to other formin proteins. Differences in the lasso/post interface and knob domain between mDia1 and HDaam1 alter the structural association between knob and post actin-binding sites, resulting in different binding angles of actin to the active site. The angle deviation induces different levels of strain on the actin fibers, thus leading to different rates of processive capping for mDia1 and HDaam1 (Lu, 1258).