Actin-binding Domain Of Utrophin Research Articles

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Binding to F-actin guides cadherin cluster assembly, stability, and movement

The cadherin extracellular region produces intercellular adhesion clusters through trans- and cis-intercadherin bonds, and the intracellular region connects these clusters to the cytoskeleton. To elucidate the interdependence of these binding events, cadherin adhesion was reconstructed from the minimal number of structural elements. F-actin-uncoupled adhesive clusters displayed high instability and random motion. Their assembly required a cadherin cis-binding interface. Coupling these clusters with F-actin through an α-catenin actin-binding domain (αABD) dramatically extended cluster lifetime and conferred direction to cluster motility. In addition, αABD partially lifted the requirement for the cis-interface for cluster assembly. Even more dramatic enhancement of cadherin clustering was observed if αABD was joined with cadherin through a flexible linker or if it was replaced with an actin-binding domain of utrophin. These data present direct evidence that binding to F-actin stabilizes cadherin clusters and cooperates with the cis-interface in cadherin clustering. Such cooperation apparently synchronizes extracellular and intracellular binding events in the process of adherens junction assembly.

Utrophin ABD binds to F-actin in an open conformation

Structural analyses of actin binding regions comprising tandem calponin homology domains alone and when bound to F-actin have revealed a number of different conformations with calponin homology domains in ‘open’ and ‘closed’ positions. In an attempt to resolve these issues we have examined the properties of the utrophin actin binding domain in open and closed conformations in order to verify the conformation when bound to F-actin. Locking the actin binding domain in a closed conformation using engineered cysteine residues in each calponin homology domain reduced the affinity for F-actin without affecting the stoichiometry furthermore differential scanning calorimetry experiments revealed a reduction in melting temperature on binding to actin. The data suggest the amino-terminal utrophin actin binding domain is in an open conformation in solution and when bound to F-actin.

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.

Acetylcholine Receptor Clustering Is Required for the Accumulation and Maintenance of Scaffolding Proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.

The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins.

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including α-actinin, β-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.

Biochemical characterisation of the actin-binding properties of utrophin.

Utrophin is a large ubiquitously expressed cytoskeletal protein that is important for maturation of vertebrate neuromuscular junctions. It is highly homologous to dystrophin, the protein defective in Duchenne and Becker muscular dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of members of the spectrin superfamily of proteins. We have determined the actin-binding properties of this utrophin domain and investigated its binding site on F-actin. An F-actin cosedimentation assay confirmed that the domain binds more tightly to beta-F-actin than to alpha-F-actin and that the full-length utrophin domain binds more tightly to both actin isoforms than a truncated construct, lacking a characteristic utrophin N-terminal extension. Both domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the domain in the presence of F-actin showed that the N-terminal extension was protected by binding to actin. The actin isoform dependence of utrophin binding could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding assays using myosin subfragment S1, which also binds F-actin near its N-terminus. Cross-linking studies suggested that utrophin contacts two actin monomers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the actin-binding properties of the utrophin actin-binding domain.

Disruption of the utrophin‒actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.

Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin-F-actin binding and was shown to recognize a predicted helical region at residues 13-22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.