F-actin Binding Site Research Articles

Arabidopsis VILLIN5 bundles actin filaments using a novel mechanism.

Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments.

Abstract Tu082: Novel Structural and Biochemical Effects of Nexilin Associated Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is primarily a genetic disease that results in abnormal cardiac muscle phenotypes characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Both sarcomere structural and signaling genes are associated with HCM. Among various genes, one of the implicated genes for HCM is Nexilin (NEXN). Nexilin is an F-actin binding protein widely expressed in muscle cells and localized at sarcomere and T-tubules. It is known to have a surprising role in insulin signaling. The role of Nexilin mutations is poorly studied. We hypothesized that Nexilin missense variants in genetically engineered human pluripotent stem cells (hPSCs) perturb essential cardiac metabolic signaling pathways leading to HCM. In our Indian HCM cohort, we identified seven Nexilin mutations through exome sequencing and categorized them as pathogenic per ACMG/AMP classification. A representative novel mutant in Nexilin p.Q131T was introduced in hPSC with the CRISPR-Cas9 knock-in method and was differentiated into cardiomyocytes, recapitulating patient-specific heterozygous mutation. Our data shows that Nexilin p.Q131T causes an increase in cell size and activation of fetal gene transcriptional programs in hPSC-derived cardiomyocytes. Global transcriptomics was performed to understand transcriptional changes and investigate further. We observe the significant activation of insulin-related signaling pathways, including AKT/MTOR and MAPK, confirmed by immunoblotting. Akt-mTOR and MAPK cascades are well-known to be regulated by IRS-1 signaling. Our studies show hyperactivation of these cascades, suggesting IRS-1 involvement in the pathogenesis. Nexilin and IRS-1 interaction were probed and found to be perturbed, hence causing higher activation of IRS-1 and its downstream targets. In addition, Nexilin wild type and mutant protein structure-functional analysis were performed with purified proteins. Using cryo-EM structural analysis, we identified unique F-actin binding sites for Nexilin for the first time, and the mutant p.Q131T showed decreased binding affinity to F-actin. We report Nexilin's high-resolution cryo-EM structure and a novel metabolic signaling mechanism where mutations in Nexilin cause dysregulation of IRS-1 signaling in the gene-edited cardiomyocytes, leading to HCM pathogenesis.

Cytosolic concentrations of actin binding proteins and the implications for in vivo F-actin turnover.

Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.2 µM, with approximately two-thirds in the F-actin form and one-third in the G-actin form. Cellular concentrations of ABPs range from 12.4 to 0.85 µM (Tpm1> Pfy1> Cof1> Abp1> Srv2> Abp140> Tpm2> Aip1> Cap1/2> Crn1> Sac6> Twf1> Arp2/3> Scp1). The cytosolic fractions of all ABPs are unexpectedly high (0.6-0.9) and remain so throughout the cell cycle. Based on these numbers, we speculate that F-actin binding sites are limited in vivo, which leads to high cytosolic levels of ABPs, and in turn helps drive the rapid assembly and turnover of cellular F-actin structures.

Functional comparison of full-length palladin to isolated actin binding domain.

Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline-rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F-actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full-length palladin in actin binding stoichiometry, polymerization, and interactions with G-actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.

Abstract 3612: KAT8/SIRT7-medicated fascin-K41 acetylation/deacetylation regulates tumor metastasis

Abstract Background: Although it is not expressed in the normal differentiated epithelial tissue of adult animals, fascin is highly expressed in a variety of cancers, including esophageal cancer, working as an important oncogenic protein. fascin promotes migration and invasion of cancer cells by binding F-actin to promote the formation of filopodia and invadopodia. However, it is not clear how exactly the function of fascin is systematically regulated in cancer cells. There are three regions on the surface of fascin protein that are significantly rich in positively charged lysine and arginine. These regions are now thought to be potential F-actin binding sites. We hypothesize that cancer cells may regulate the function of fascin by post-translational modifications of key amino acids in these three regions, especially by acetylation. Based on this, this study aims to identify acetyltransferases that regulate the acetylation of fascin in esophageal cancer cells and reveal the effect of acetylation on the function of fascin. Results: 1) Co-IP, pull-down, and immunofluorescence results showed that KAT8 interact with fascin and co-localize in esophageal cancer cells. 2) Mass spectrometry identification of the products of acetylation in vitro revealed that KAT8 acetylates fascin at K41 and K241. By means of Ack41-fascin specific antibodies, further analysis proved that KAT8 acetylated fascin K41. 3) Co-IP and immunofluorescence results showed that SIRT7 interact with fascin and co-localize in esophageal cancer cells, moreover, treatment of cells with SIRT7-specific inhibitor significantly increased the level of AcK41-fascin, and however, overexpressing SIRT7 reduces the level of AcK41-fascin. 4) Immunohistochemical results showed that the level of AcK41-fascin was lower in esophageal cancer tissues of patients with lymph node metastasis; Analysis of overall survival and tumor-free survival curves showed that patients with high levels of AcK41-fascin had a better prognosis. 5) Cell wound healing and transwell assay showed that fascin K41 residue mimics acetylation inhibit the movement and invasion of esophageal cancer cells. 6) Furthermore, in vitro F-actin binding assays showed that mutations mimicing acetylation of K41 inhibited the activity of fascin bunding F-actin. 7) Immunofluorescence assay showed that fascin K41 residue mimics acetylation inhibited fascin's ability to promote filopodia and invadopodia formation in esophageal cancer cells. Conclusions: 1) KAT8 is an acetyltransferase that catalyzes fascin K41 acetylation in esophageal cancer cells, while SIRT7 decides to catalyze AcK41-fascin deacetylation. 2) SIRT7-mediated deacetylation of AcK41-fascin promotes the formation of filopodia and invadopodia, which promotes the invasion and metastasis of esophageal cancer cells. (This work was supported by grants from the National Natural Science Foundation of China (No.81872372 and 81902469)) Citation Format: Da-Jia Li, Yin-Wei Cheng, Li-Yan Xu, En-Min Li. KAT8/SIRT7-medicated fascin-K41 acetylation/deacetylation regulates tumor metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3612.

Functional characterisation of the actin-depolymerising factor from the apicomplexan Neospora caninum (NcADF)

Neospora caninum is an apicomplexan parasite that causes infectious abortion in cows. As an obligate intracellular parasite, N. caninum requires a host cell environment to survive and replicate. The locomotion and invasion mechanisms of apicomplexan parasites are centred on the actin-myosin system to propel the parasite forwards and into the host cell. The functions of actin, an intrinsically dynamic protein, are modulated by actin-binding proteins (ABPs). Actin-depolymerising factor (ADF) is a ubiquitous ABP responsible for accelerating actin turnover in eukaryotic cells and is one of the few known conserved ABPs from apicomplexan parasites. Apicomplexan ADFs have nonconventional properties compared with ADF/cofilins from higher eukaryotes. In the present paper, we characterised the ADF from N. caninum (NcADF) using computational and in vitro biochemical approaches to investigate its function in rabbit muscle actin dynamics. Our predicted computational tertiary structure of NcADF demonstrated a conserved structure and phylogeny with respect to other ADF/cofilins, although certain differences in filamentous actin (F-actin) binding sites were present. The activity of recombinant NcADF on heterologous actin was regulated in part by pH and the presence of inorganic phosphate. In addition, our data suggest a comparatively weak disassembly of F-actin by NcADF. Taken together, the data presented herein represent a contribution to the field towards the understanding of the role of ADF in N. caninum and a comparative analysis of ABPs in the phylum Apicomplexa.

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1.

EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.

Interactions between the Microtubule Binding Protein EB1 and F-Actin

Many cellular processes including cell division and cell migration require coordination between the actin and microtubule (MT) cytoskeletons. This coordination is as-yet poorly understood, but proteins such as formins and IQGAP1 are known to be involved. We show that the MT binding protein EB1 (end-binding protein 1), a key regulator of MT dynamics, can bind directly to filamentous actin (F-actin) F-actin. We determined that the EB1:F-actin interaction is salt sensitive and weak under physiological salt concentrations but might be relevant in contexts where the local concentration of actin is high. Using bioinformatics and mutagenesis, we found that the EB1:F-actin binding site partially overlaps the well-characterized EB1:MT binding interface. Congruently, competition experiments indicate that EB1 can bind to F-actin or MTs but not both simultaneously. These observations suggest that EB1:F-actin interactions may negatively regulate EB1:MT interactions, and we speculate that this interaction may assist cells in differentially regulating MT stability in the actin-rich cortex as opposed to the cell interior.

Self-Organization of Actomyosin Networks Attached to Artificial Membranes

The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments.In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments.Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement.

Assessing the Functional Role of Titin N2A-PEVK Region in Active Muscle Contraction

Muscle contraction is an interplay of cyclic interactions between the contractile proteins myosin and actin driven by ATP turnover. Despite extensive studies and clinical significance of muscle, there are still unanswered questions about active muscle contraction especially eccentric contractions. The sliding filament theory explains many key aspects of muscle contraction but does not adequately explain short range stiffness, enhancement of force with stretch and depression of force with shortening. We aim to provide a new insight in the mechanism of muscle contraction by further elucidating the role of titin in active muscles. Recent studies have proposed two models for the role of titin in active muscles. In model one, PEVK region of titin binds to actin upon Ca+2 activation. In the second model, N2A region of titin binds to actin upon calcium influx. In this study, we are trying to understand the molecular mechanism of active muscle contraction by identifying the F-actin binding site on titin. Using an actin sedimentation assay, we have demonstrated that a recombinantly expressed truncation of mouse titin consisting of the N2A region and the first one hundred amino acids of the PEVK region binds F-actin, validating our system. We are currently working to determine whether the binding is calcium dependent and to determine the binding constants for this association. We are also testing smaller constructs of N2A and PEVK to identify the specific amino acid sequence responsible for binding. Our studies will provide a new dimension to the existing sliding filament theory and fill gaps in the molecular mechanism of active muscle contraction and movement.

Inositol(1,4,5)P3 3-kinase isoenzymes: Catalytic properties and importance of targeting to F-actin to understand function.

Inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) 3-kinases (Itpks) catalyze the phosphorylation of inositol(1,4,5)trisphosphate into inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4). Three isoenzymes Itpka/b and c have been identified in human, rat and mouse. They share a catalytic domain relatively well conserved at the C-terminal end and a quite isoenzyme specific regulatory domain at the N-terminal end of the protein. Activity determined in cell homogenates with Ins(1,4,5)P3 and ATP as substrate is generally very low compared to Ins(1,4,5)P3 5-phosphatase, except in a few tissues such as brain, testis, thymus or intestine. Activity is very much Ca(2+) sensitive and increased in the presence of Ca(2+)/calmodulin (CaM) as compared to EGTA alone. When challenged after receptor activation, activity could be further activated several fold, e.g. in rat brain cortical slices stimulated by carbachol or in human astrocytoma cells stimulated by purinergic agonists. Two of the three isoenzymes show an unexpected cytoskeletal localization for Itpka/b or at the leading edge for Itpkb. This is explained by the presence of an F-actin binding site at the N-terminal part of the two isoenzymes. This interaction confers to Itpka the properties of an F-actin bundling protein with two major consequences: i) it can reorganize the cytoskeletal network, particularly in dendritic spines, and ii) can provide an opportunity for Ins(1,3,4,5)P4 to act very locally as second messenger.

Drebrin contains a cryptic F-actin–bundling activity regulated by Cdk5 phosphorylation

Drebrin is an actin filament (F-actin)-binding protein with crucial roles in neuritogenesis and synaptic plasticity. Drebrin couples dynamic microtubules to F-actin in growth cone filopodia via binding to the microtubule-binding +TIP protein EB3 and organizes F-actin in dendritic spines. Precisely how drebrin interacts with F-actin and how this is regulated is unknown. We used cellular and in vitro assays with a library of drebrin deletion constructs to map F-actin binding sites. We discovered two domains in the N-terminal half of drebrin-a coiled-coil domain and a helical domain-that independently bound to F-actin and cooperatively bundled F-actin. However, this activity was repressed by an intramolecular interaction relieved by Cdk5 phosphorylation of serine 142 located in the coiled-coil domain. Phospho-mimetic and phospho-dead mutants of serine 142 interfered with neuritogenesis and coupling of microtubules to F-actin in growth cone filopodia. These findings show that drebrin contains a cryptic F-actin-bundling activity regulated by phosphorylation and provide a mechanistic model for microtubule-F-actin coupling.

GMF Severs Actin-Arp2/3 Complex Branch Junctions by a Cofilin-like Mechanism

Branched actin filament networks driving cell motility, endocytosis, and intracellular transport are assembled in seconds by the Arp2/3 complex and must be equally rapidly debranched and turned over. One of the only factors known to promote debranching of actin networks is the yeast homolog of glia maturation factor (GMF), which is structurally related to the actin filament-severing protein cofilin. However, the identity of the molecular mechanism underlying debranching and whether this activity extends to mammalian GMF have remained open questions. Using scanning mutagenesis and total internal reflection fluorescence microscopy, we show that GMF depends on two separate surfaces for debranching. One is analogous to the G-actin and F-actin binding site on cofilin, but we show using fluorescence anisotropy and chemical crosslinking that it instead interacts with actin-related proteins in the Arp2/3 complex. The other is analogous to a second F-actin binding site on cofilin, which in GMF appears to contact the first actin subunit in the daughter filament. We further show that GMF binds to the Arp2/3 complex with low nanomolar affinity and promotes the open conformation. Finally, we show that this debranching activity and mechanism are conserved for mammalian GMF. GMF debranches filaments by a mechanism related to cofilin-mediated severing, but in which GMF has evolved to target molecular junctions between actin-related proteins in the Arp2/3 complex and actin subunits in the daughter filament of the branch. This activity and mechanism are conserved in GMF homologs from evolutionarily distant species.

The Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)

Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: Genes, expression patterns and protein interactions of two potential moonlighting proteins

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon–intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory–secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host–parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host–parasite interplay.

An MBoC Favorite: Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site

The ezrin, radixin, and moesin (ERM) proteins have emerged as important regulators of the cortical actin cytoskeleton, particularly in the apical domain of polarized epithelial cells. In this paper (Gary and Bretscher, 1995), which has been cited more than 250 times since publication, the authors showed that head-to-tail binding regulates interactions with actin and other binding partners at the cell cortex. Intramolecular head-to-tail binding has subsequently been shown to be critical for regulation of ERM proteins, as well as for the closely related protein Merlin, a component of the Hippo growth control pathway. ERM folding has also served as a model for autoinhibition of a number of other cortical cytoskeletal proteins. Interestingly, although the authors’ data suggested the existence of both intramolecular and intermolecular interactions, little is known about the importance of the latter in living cells, suggesting that the full importance of this work remains to be discovered.

Solution structure and dynamics of ADF from Toxoplasma gondii

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹⁵N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.

Molecular cloning and characterization of an actin- depolymerizing factor gene in Hevea brasiliensis

Actin-depolymerizing factor (ADF) plays important roles in regulating actin dynamics by maintaining the optimum equilibrium between unpolymerized actin molecules and assembled actin filaments in different cellular processes. In this study, the first ADF gene in Hevea brasiliensis designated as HbADF, was isolated. The HbADF contained an open reading frame (ORF) encoding 139 amino acids. The deduced HbADF showed high identities to plants ADF proteins. Besides a conserved ADF domain, HbADF also contained putative actin and specific F-actin binding sites, phosphorylation site and possible CAM (calmodulin) combining region. The phylogenetic analysis indicated that HbADF was clustered in the subclass I. Being consistent with phylogenetic result, the expression of HbADF was constitutive. The HbADF transcripts were upregulated by ethephon and wounding treatments; whereas, HbADF was firstly induced, and then gradually downregulated by jasmonic acid. The expression profiles and characterizations of HbADF suggested that HbADF might be associated with latex regeneration and flow in H. brasiliensis.