Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin.

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs act as actin-activated NC toxins. Here, we explore the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) instead of filamentous (F-actin) actin. Vn-ExoY exhibits moderate binding affinity to free or profilin-bound G-actin but can capture the G-actin:profilin complex, preventing its spontaneous or VASP- or formin-mediated assembly at F-actin barbed ends in vitro. This mechanism may prolong the activated cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. We present a series of high-resolution crystal structures of nucleotide-free, 3'-deoxy-ATP- or 3'-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP, revealing that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of NC toxins. Substrate binding induces a large, previously-unidentified, closure of their NBP, confining catalytically important residues and metal cofactors around the substrate, and facilitating the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. We validate critical residues for both the purinyl and pyrimidinyl cyclase activity of NC toxins in Vn-ExoY and its distantly-related ExoY from Pseudomonas aeruginosa, which specifically interacts with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins offer new perspectives for the specific inhibition of class II bacterial NC enzymes.

Bacterial Nucleotidyl Cyclases Activated by Calmodulin or Actin in Host Cells: Enzyme Specificities and Cytotoxicity Mechanisms Identified to Date.

Many pathogens manipulate host cell cAMP signaling pathways to promote their survival and proliferation. Bacterial Exoenzyme Y (ExoY) toxins belong to a family of invasive, structurally-related bacterial nucleotidyl cyclases (NC). Inactive in bacteria, they use proteins that are uniquely and abundantly present in eukaryotic cells to become potent, unregulated NC enzymes in host cells. Other well-known members of the family include Bacillus anthracis Edema Factor (EF) and Bordetella pertussis CyaA. Once bound to their eukaryotic protein cofactor, they can catalyze supra-physiological levels of various cyclic nucleotide monophosphates in infected cells. Originally identified in Pseudomonas aeruginosa, ExoY-related NC toxins appear now to be more widely distributed among various γ- and ß-proteobacteria. ExoY-like toxins represent atypical, poorly characterized members within the NC toxin family. While the NC catalytic domains of EF and CyaA toxins use both calmodulin as cofactor, their counterparts in ExoY-like members from pathogens of the genus Pseudomonas or Vibrio use actin as a potent cofactor, in either its monomeric or polymerized form. This is an original subversion of actin for cytoskeleton-targeting toxins. Here, we review recent advances on the different members of the NC toxin family to highlight their common and distinct functional characteristics at the molecular, cytotoxic and enzymatic levels, and important aspects that need further characterizations.

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin.

Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.

Cordon-Bleu uses WH2 domains as multifunctional dynamizers of actin filament assembly.

Cordon-Bleu is, like Spire, a member of the growing family of WH2 repeat proteins, which emerge as versatile regulators of actin dynamics. They are expressed in morphogenetic and patterning processes and nucleate actin assembly in vitro. Here, we show that Cordon-Bleu works as a dynamizer of actin assembly by combining many properties of profilin with weak filament nucleating and powerful filament severing activities and sequestration of ADP-actin, which altogether generate oscillatory polymerization kinetics. A short lysine-rich sequence, N-terminally adjacent to the three WH2 domains, is required for nucleation and severing. In this context, nucleation requires only one WH2 domain, but filament severing requires two adjacent WH2 domains. A model integrating the multiple activities of Cordon-Bleu and quantitatively fitting the multiphasic polymerization curves is derived. Hence, with similar structural organization of WH2 repeats, Cordon-Bleu and Spire display different functions by selecting different sets of the multifunctional properties of WH2 domains.

How a single residue in individual ß-thymosin/WH2 domains controls their functions in actin assembly.

ß-Thymosin (ßT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-ß4, or enhance motility by directing polarized filament assembly like Ciboulot ßT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-ß4, Ciboulot, TetraThymosinß and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-ß4. Functionally different ßT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long ßT and WH2 domains. The results open perspectives for elucidating the functions of ßT/WH2 domains in other modular proteins.

Small-angle X-ray solution scattering study of the multi-aminoacyl-tRNA synthetase complex reveals an elongated and multi-armed particle.

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Interactions of isolated C-terminal fragments of neural Wiskott-Aldrich syndrome protein (N-WASP) with actin and Arp2/3 complex.

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 complex. The VCA·actin·Arp2/3 entity associates with a mother filament to form a branched junction from which a daughter filament is initiated. The number and function of WH2-bound actin(s) in the branching process are not known, and the stoichiometry of the VCA·actin·Arp2/3 complex is debated. We have expressed the tandem WH2 repeats of N-WASP, either alone (V) or associated with the C (VC) and CA (VCA) extensions. We analyzed the structure of actin in complex with V, VC, and VCA using protein crystallography and hydrodynamic and spectrofluorimetric methods. The partial crystal structure of the VC·actin 1:1 complex shows two actins in the asymmetric unit with extensive actin-actin contacts. In solution, each of the two WH2 domains in V, VC, and VCA binds G-actin in 1:2 complexes that participate in barbed end assembly. V, VC, and VCA enhance barbed end depolymerization like profilin but neither nucleate nor sever filaments, in contrast with other WH2 repeats. VCA binds the Arp2/3 complex in a 1:1 complex even in the presence of a large excess of VCA. VCA·Arp2/3 binds one actin in a latrunculin A-sensitive fashion, in a 1:1:1 complex, indicating that binding of the second actin to VCA is weakened in the ternary complex.

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the beta-phosphates in the gamma-phosphate-binding site.

Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells.

ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor.

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.

ExoY, an actin-activated nucleotidyl cyclase toxin from P. aeruginosa: A minireview.

ExoY is one of four well-characterized Pseudomonas aeruginosa type 3 secretion system (T3SS) effectors. It is a nucleotidyl cyclase toxin that is inactive inside the bacteria, but becomes potently activated once it is delivered into the eukaryotic target cells. Recently, filamentous actin was identified as the eukaryotic cofactor that stimulates specifically ExoY enzymatic activity by several orders of magnitude. In this review, we discuss recent advances in understanding the biochemistry of nucleotidyl cyclase activity of ExoY and its regulation by interaction with filamentous actin.

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin.

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

Structure, function, and evolution of the beta-thymosin/WH2 (WASP-Homology2) actin-binding module.

beta-thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

Unique GMP-binding site in Mycobacterium tuberculosis guanosine monophosphate kinase.

Bacterial nucleoside monophosphate (NMP) kinases, which convert NMPs to nucleoside diphosphates (NDP), are investigated as potential antibacterial targets against pathogenic bacteria. Herein, we report the biochemical and structural characterization of GMP kinase from Mycobacterium tuberculosis (GMPKMt). GMPKMt is a monomer with an unusual specificity for ATP as a phosphate donor, a lower catalytic efficiency compared with eukaryotic GMPKs, and it carries two redox-sensitive cysteines in the central CORE domain. These properties were analyzed in the light of the high-resolution crystal structures of unbound, GMP-bound, and GDP-bound GMPKMt. The latter structure was obtained in both an oxidized form, in which the cysteines form a disulfide bridge, and a reduced form which is expected to correspond to the physiological enzyme. GMPKMt has a modular domain structure as most NMP kinases. However, it departs from eukaryotic GMPKs by the unusual conformation of its CORE domain, and by its partially open LID and GMP-binding domains which are the same in the apo-, GMP-bound, and GDP-bound forms. GMPKMt also features a unique GMP binding site which is less close-packed than that of mammalian GMPKs, and in which the replacement of a critical tyrosine by a serine removes a catalytic interaction. In contrast, the specificity of GMPKMt for ATP may be a general feature of GMPKs because of an invariant structural motif that recognizes the adenine base. Altogether, differences in domain dynamics and GMP binding between GMPKMt and mammalian GMPKs should reveal clues for the design of GMPKMt-specific inhibitors.

Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase.

Guanosine monophosphate kinases (GMPKs), which catalyze the phosphorylation of GMP and dGMP to their diphosphate form, have been characterized as monomeric enzymes in eukaryotes and prokaryotes. Here, we report that GMPK from Escherichia coli (ecGMPK) assembles in solution and in the crystal as several different oligomers. Thermodynamic analysis of ecGMPK using differential scanning calorimetry shows that the enzyme is in equilibrium between a dimer and higher order oligomers, whose relative amounts depend on protein concentration, ionic strength, and the presence of ATP. Crystallographic structures of ecGMPK in the apo, GMP and GDP-bound forms were solved at 3.2A, 2.9A and 2.4A resolution, respectively. ecGMPK forms a hexamer with D3 symmetry in all crystal forms, in which the two nucleotide-binding domains are able to undergo closure comparable to that of monomeric GMPKs. The 2-fold and 3-fold interfaces involve a 20-residue C-terminal extension and a sequence signature, respectively, that are missing from monomeric eukaryotic GMPKs, explaining why ecGMPK forms oligomers. These signatures are found in GMPKs from proteobacteria, some of which are human pathogens. GMPKs from these bacteria are thus likely to form the same quaternary structures. The shift of the thermodynamic equilibrium towards the dimer at low ecGMPK concentration together with the observation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be the active species at physiological enzyme concentration.

Mutations in actin used for structural studies partially disrupt ß-thymosin/WH2 domains interaction.

Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model ß-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both ß-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins.

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.

Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties.

More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of ß-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

Structural features and interfacial properties of WH2, ß-thymosin domains and other intrinsically disordered domains in the regulation of actin cytoskeleton dynamics.

Many actin-binding proteins (ABPs) use complex multidomain architectures to integrate and coordinate multiple signals and interactions with the dynamic remodeling of actin cytoskeleton. In these proteins, small segments that are intrinsically disordered in their unbound native state can be functionally as important as identifiable folded units. These functional intrinsically disordered regions (IDRs) are however difficult to identify and characterize in vitro. Here, we try to summarize the state of the art in understanding the structural features and interfacial properties of IDRs involved in actin self-assembly dynamics. Recent structural and functional insights into the regulation of widespread, multifunctional WH2/ß-thymosin domains, and of other IDRs such as those associated with WASP/WAVE, formin or capping proteins are examined. Understanding the functional versatility of IDRs in actin assembly requires apprehending by multiple structural and functional approaches their large conformational plasticity and dynamics in their interactions. In many modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in interdomain and protein-protein interfaces. They thus control multiple conformational transitions between the inactive and active states or between various active states of multidomain ABPs, and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics.

Control of actin assembly by the WH2 domains and their multifunctional tandem repeats in Spire and Cordon-Bleu.

The WASP-homology 2 (WH2) domain is a 5-kDa actin-binding protein module that attracts increasing interest by its multifunctional regulation of actin dynamics in motile and morphogenetic processes. Identified by a short consensus sequence LKKT/V originally found in the actin-sequestering ß-thymosin peptides, the ßT/WH2 domains are inserted in a large number of proteins, in particular, the WASP proteins involved in cell protrusions. WH2 are found in tandem repeats in proteins involved in early development and axis-patterning processes, like Spire and Cordon-Bleu. These intrinsically disordered proteins regulate actin assembly in an adaptive and versatile fashion by a fine control of local interaction dynamics within the WH2-actin complex. Versatility is amplified by the protein environment in which the WH2 domain is inserted and by synergy with other adjacent actin-binding modules. Multifunctional activities emerge in WH2 repeats, including filament nucleation, dramatic severing, and barbed end capping or tracking. WH2 domains thus are instrumental in designing customized actin regulators.