The IMD of MIM is an all α-helical structure, which dimerizes to form a twisted ellipsoid ~183 Å in length, with a large cavity in the middle (). Despite low sequence similarity the structures of the IMDs of MIM and IRSp53 (Millard et al., 2005
) are generally similar. The loop following α-helix 3 of MIM’s IMD forms a “flap” that covers the so-called “signature sequence” of the IMD, a conserved and charged sequence that is conspicuously buried in the structure ().
While we were able to confirm that the IMD binds F-actin with ~17 μM affinity (), we found that the symmetric patches of basic amino acids at the distal ends of the dimer () play only a limited role in this interaction (). Furthermore, we did not observe any bundling activity for the IMD (), which would have been consistent with the symmetric ends of the dimer being involved in actin binding. If as previously suggested (Yamagishi et al., 2004
) MIM is an actin bundling protein, this function may require other parts of the molecule that lie outside the IMD. Gonzalez-Quevedo et al
. (Gonzalez-Quevedo et al., 2005
) reached a similar conclusion by studying various fragments of MIM. They showed that most of the bundling activity could be restored by a construct comprising amino acids 1 to 408 of MIM. Another possibility is that bundling is regulated (or potentiated) in vivo
by still unknown factors.
The IMD of IRSp53 interacts with Rac, possibly functioning as an intermediate for the activation of WAVE, which is recruited by the SH3 domain of IRSp53 (Miki et al., 2000
). Similarly, the IMD of MIM has been shown to bind and activate Rac, suggesting that MIM could link Rac to effector proteins involved in lamellipodia formation, such as WAVE (Bompard et al., 2005
). The structural basis for the Rac-IMD interaction is unknown. Interestingly, the structure of the IMD resembles that of the BAR domain, which also binds small GTPases (Habermann, 2004
). The crystal structures of various BAR-domain proteins, including arfaptin (Tarricone et al., 2001
), amphiphysin (Peter et al., 2004
) and endophilin (Weissenhorn, 2005
) have been determined. Although the BAR domain is curved and the IMD is relatively straight, the two folds superimpose remarkably well in the middle section, where the two subunits that conform these two domains overlap (). It is via
this well-overlapping middle section that the binding of small GTPases appears to take place. Indeed, the structure of arfaptin was also determined bound to Rac (Tarricone et al., 2001
). One molecule of Rac sits at the midpoint of the arfaptin BAR dimer. It is likely that the IMDs of MIM and IRSp53 bind Rac in a similar fashion, as illustrated by a superimposition of the structures of MIM and arfaptin-Rac (). Note, however, that this superimposition does not represent an accurate model of the interaction, since there is no obvious sequence similarity between the IMD and BAR domains and local changes are likely.
The binding of Rac and actin by the IMD of MIM appear to be mutually exclusive (Bompard et al., 2005
). Although this study did not determine the total extent of the actin-binding interface, the lack of bundling activity () and the fact that the distal ends of the IMD dimer do not constitute a major actin-binding site () would suggest that the middle section of the IMD dimer also participates in actin binding. As suggested by the analogy with the BAR domain, the binding of Rac may also involve the middle section of the IMD dimer (), possibly explaining why actin and Rac bind in a mutually exclusive manner.
We have stressed here the striking resemblance between the IMD and BAR folds, including their shared ability to bind small GTPases. In addition, both domains present similar clusters of positively charged amino acids (), which in the BAR domain coincide with the concave surface of the dimer and are involved in phospholipid membrane binding (Peter et al., 2004
). The most noticeable difference between the two folds is that the IMD forms relatively straight dimers (Millard et al., 2005
), whereas the BAR domain forms curved “banana-shaped” dimers (Peter et al., 2004
; Tarricone et al., 2001
; Weissenhorn, 2005
). However, the curvature of the BAR domain varies from protein to protein (arfaptin > amphiphysin > endophilin), which may facilitate the binding to membranes with different curvatures. The IMD was discovered independently and due to the lack of sequence similarity was not originally considered a member of the BAR domain family (Yamagishi et al., 2004
). A comparison of the structures of the IMD and BAR domains would now suggest that the two domains are not only structurally but also functionally related to each other (). Indeed, it was recently reported that like the BAR domain the IMD also binds membranes and that this function is mediated by the clusters of basic amino acids at the distal ends of the dimer (Suetsugu et al., 2006
). Interestingly, the directionality of membrane deformation by the IMD (outward) was found to be opposite to that produced by the BAR domain (inward). The structures may provide an explanation for this observation since the concave and positively charged surface implicated in membrane binding in the BAR domain adopts a somewhat convex shape in the IMD (). Therefore, the evidence to date suggests that the IMD is a multifunctional module, linking the actin cytoskeleton to the formation of membrane protrusions by direct interactions with both F-actin and membranes, all under the control of the small GTPase Rac.
The WH2 of MIM interacts with all four subdomains of actin (). It consists of an N-terminal amphiphilic α-helix that binds in the cleft between actin subdomains 1 and 3 and a C-terminal extended region that binds along the actin surface and the nucleotide cleft reaching the top of actin subdomains 2 and 4. Note that the end of this WH2 coincides with the C-terminus of the MIM protein. The prototypical WH2 found among WASP-family proteins tends to be shorter (), and presents little or no interactions with actin after the LKKT sequence (Chereau et al., 2005
We demonstrated here that certain isoforms of IRSp53 present a C-terminal WH2 that binds actin with similar affinity to that of MIM’s WH2, further extending the relationship between these two actin-cytoskeleton scaffolding proteins. WH2 is the smallest actin-binding motif known. Based on their sequences and structures, we have identified two types of WH2s; long and short (Chereau et al., 2005
). Short WH2s consist solely of the N-terminal α-helix and the LKKT-related sequence (for example WASP’s WH2, ). Long WH2s present an additional ~10 amino acids at the C-terminus. The extra amino acids of long WH2s share sequence similarity with T β4 and make similar contacts with actin (Irobi et al., 2004
), supporting a previously proposed relationship between the WH2 and T β families (Paunola et al., 2002
). However, it remains unclear whether the extra amino acids of long WH2s play any specific role, since they don’t seem to contribute significantly to the actin binding affinity nor the nucleotide exchange inhibition by actin (Chereau et al., 2005
What is the role of WH2 in MIM and IRSp53? WH2 could serve two possible functions: recruit actin monomers, or recruit a protein to a specific actin cytoskeletal network. Actin filament nucleation and elongation factors, including WASP, Ena/VASP and spire, form the main group of WH2-containing proteins. These proteins present short WH2s, typically positioned C-terminal to Pro-rich sequences (). In WASP WH2 is followed by the central (or C) region that binds one of the subunits of Arp2/3 complex, whereas in VASP WH2 is known as the G-actin-binding domain (GAB) and is followed by the F-actin-binding domain (FAB). The C region of WASP and the FAB domain of VASP are related to each other, and both constitute specialized forms of WH2 (Chereau and Dominguez, 2006
). Spire, on the other hand, contains four WH2s in tandem (Quinlan et al., 2005
). We have proposed that in these proteins WH2 becomes involved in nucleation and elongation by forming nuclei for actin assembly and by mediating the incorporation of profilin-actin at the barbed end of growing filaments (Chereau and Dominguez, 2006
; Chereau et al., 2005
). So far, we have identified long WH2s in actobindin, WIP, MIM (Chereau et al., 2005
), and now in IRSp53. It appears that in MIM and IRSp53 WH2 occurs within a different domain organization than in most cytoskeletal proteins (). Thus, in MIM and IRSp53 WH2 is found in isolation at the C-terminal end; i.e.
not immediately preceded by Pro-rich sequences nor followed by other WH2s (or WH2-related sequences). Unlike the actin monomer-trapping molecule T β4 and the nucleation-elongation factors described above, MIM and IRSp53 function as scaffolding proteins. It is therefore likely that WH2 helps recruit MIM and IRSp53, as well as their multiple binding partners, to specific cytoskeletal networks. Consistent with this idea, images of cells overexpressing full-length MIM show a significant loss of stress fibers (Gonzalez-Quevedo et al., 2005
; Mattila et al., 2003
; Woodings et al., 2003
), but this effect appears diminished for MIM constructs lacking the WH2 region (Bompard et al., 2005
; Gonzalez-Quevedo et al., 2005
What is the spatial relationship between the IMD and WH2 domains? Hydrophobic cluster analysis (Callebaut et al., 1997
) suggests that the region sandwiched in between the IMD and WH2 of MIM is mostly unstructured, with only two segments with predicted globular or inducible folding (Figure S2
). Given these characteristics and the antiparallel organization of the IMD dimer, the two WH2s could be located far apart from each other in the protein, which would imply a lack of communication between them. More likely, however, the various domains of MIM and IRSp53 fold back into a more compact structure, possibly mediated by auto-regulatory interactions involving the IMD and other parts of the molecule.