Autoinhibition of mDial

A number of studies have begun to unravel the mechanism of mDial autoinhibition. Early cellular studies utilizing truncated mDial constructs demonstrated the involvement of both N-terminal and C-terminal domains in the regulation of mDia-mediated actin filament assembly (Watanabe et al. 1997; Tominaga et al. 2000; Alberts 2001). Subsequently, these regulatory activities were discovered to be the result of an interaction between a domain in the amino terminus of mDial termed the diaphanous inhibitor domain (DID) and a domain adjacent to the C-terminal FH2 domain termed the diaphanous autoregulatory domain (DAD) (Fig. 3). Biophysical studies demonstrated binding of the DID to the DAD with sub-micromolar affinity (Lammers et al. 2005; Li and Higgs 2005), and introduction of a recombinant peptide comprising the DID is sufficient to inhibit FH2-DAD-induced

Fig. 3 Schematic representation of autoinhibition of mDial (mDial is shown as a monomer for clarity). Interaction of the diaphanous inhibitor domain (DID) with the diaphanous autoregulatory domain (DAD) maintains mDial in a conformation that renders the protein unable to nucleate actin filaments via the FH2 domain (a). Binding of activated Rho to the GTPase binding domain (GBD), which partially overlaps with DID, displaces the DAD, leading to the relief of autoinhibition (b)

Fig. 3 Schematic representation of autoinhibition of mDial (mDial is shown as a monomer for clarity). Interaction of the diaphanous inhibitor domain (DID) with the diaphanous autoregulatory domain (DAD) maintains mDial in a conformation that renders the protein unable to nucleate actin filaments via the FH2 domain (a). Binding of activated Rho to the GTPase binding domain (GBD), which partially overlaps with DID, displaces the DAD, leading to the relief of autoinhibition (b)

actin polymerization (Li and Higgs 2005). Structural studies have revealed extensive hydrophobic contacts between a helix within the core DAD and a number of the armadillo-repeats contained within the DID (Lammers et al. 2005). Although the autoinhibitory domains of mDial are distinct in both amino acid sequence and three-dimensional structure from those found in N-WASP and Pakl, the general strategy of autoinhibition involving an interaction between autoregulatory domains and domains within the catalytic portion of the protein is shared among all three proteins (compare Figs. la, 2b, and 3).

Also akin to N-WASP and Pakl, DRFs contain an amino-terminal region that mediates the interaction between these proteins and Rho GTPases. The GTPase-bind-ing sites of DRFs are less well defined, in general, than for N-WASP and Pakl (Rivero et al. 2005). However, conserved CRIB-type GTPase binding domains are observed in mDia family members (Peng et al. 2003). mDial has been shown to interact with Rho A, B, and C, but not Rac or Cdc42 (Watanabe et al. l997, l999). Additionally, other mDia isoforms have been shown to interact with other Rho GTPases (Alberts et al. l998; Gasman et al. 2003; Peng et al. 2003; Yasuda et al. 2004). The mDial GBD partially overlaps with the autoregulatory region of the protein (Fig. 3), and a number of studies demonstrate that binding of activated Rho to mDial fragments containing the GBD antagonizes the DID/DAD interaction by actively displacing the DAD from the DID (Watanabe et al. l997; Lammers et al. 2005; Li and Higgs 2005; Rose et al. 2005). The functional relevance of this disruption is highlighted by data demonstrating that Rho partially relieves the inhibitory effects of the DID on mDia-mediated actin polymerization (Li and Higgs 2003).

Although much has been learned concerning the autoregulatory cycle of mDial, the molecular details of Rho-mediated relief of autoinhibition are not entirely clear. For example, binding of activated Rho or the DAD to DID-containing fragments is mutually exclusive, yet they do not bind to completely overlapping DID sequences (Lammers et al. 2005; Rose et al. 2005). Unlike what is observed in N-WASP and Pakl, release of mDial from the autoinhibited state does not appear to involve conformational changes within the autoregulatory domain, as the structure of DID fragments are not significantly different when examined alone (Otomo et al. 2005), in a complex with active Rho (Rose et al. 2005), or when bound to the DAD (Lammers et al. 2005). Currently there is no crystal structure available for the DAD alone, precluding any comparisons between the conformation of the DAD in the unbound state and the conformation of the DAD complexed with the DID. Comparison of the structures of DID-containing fragments complexed with either the DAD or RhoC led Lammers et al. to suggest a theoretical ternary Rho/DID/ DAD complex in which residues within the DAD (ll79-ll87) and RhoC (64-67) sterically and electrostatically repel each other, mediating the release of the DAD from the DID (Lammers et al. 2005). However, further study is required to demonstrate the true role of these residues in the proposed interactions.

Despite using structurally distinct domains, the regulatory mechanism of mDial is remarkably similar to that observed for N-WASP and Pakl. It is therefore conceivable that, much like N-WASP, mDial may be susceptible to inhibition by allosteric inhibition. In the following sections, we will discuss the relevance of such an inhibitor and propose screening strategies that could be used to identify small-molecule mDial inhibitors that target this protein by an allosteric mechanism.

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