Here, we present a microscale thermophoresis mst based assay for in vitro assessment of actin polymerization. Aug 05, 2008 the initiation of actin polymerization in cells requires actin filament nucleators. Cd study of the actin dnase i complex sciencedirect. Generally, it is considered that unfolded actin aggregates do not bind dnasei and therefore, dnasei inhibition is often used to infer the proportion of folded and unfolded actin within a sample 20. The bright green fluorescent alexa fluor 488 dnase i selectively binds to g actin globular and can be used for the detection of unpolymerized actin in fixed cells. However, since actinbound dnase i is enzymatically inactive, the dnase actin complex might be a storage form. The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease i both in the atp and adp forms have been determined byo xray analysis at an. Actin subdomain 2 contains the dloop recognized by dnase i. In particular, we found that rapid apical actin polymerization is linked to pollen tube growth zhang et al. The ability of actin to inhibit dnase i activity reflects the number of available dnase i binding sites in a sample of actin. We demonstrate that a ribose modified analogue of atp, tnpatp, can exchange with a resident nucleotide in f actin, but fails to bind to g actin. Dnase i complex article pdf available in nature 3476288. Engineering actinresistant human dnase i for treatment of. Nucleotide exchange gactin either mg or caform was freed from excess nucleotide by gel filtration in atpfree g.
This has been used to develop a simple, spectrophotometric assay for the quantification of unpolymerized and filamentous actin which can be applied even to crude cell extracts 911. Actin subdomains 1 and 2 small lobe and subdomains 3 and 4 large lobe are indicated. Dnase i actin complex formation is studied in the presence of different anti actin antibody populations. Structural basis for the slow dynamics of the actin filament. Based on the knowledge that actin is the natural inhibitor of dnase i, 15 we next examined the interaction of gelsolinactindnase i in the actin complex. This fragment was modified with sal1 linkers and cloned into p8 cat, a derivative of the pembl plasmid dente et al. However, since actin bound dnase i is enzymatically inactive, the dnase actin complex might be a storage form of dnase i that prevents damage of the genetic information. In the profilinactin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4. The binding activity is widely distributed in a variety of cells and tissues. Subdomain location of mutations in cardiac actin correlate. Gelsolin regulates cardiac remodeling after myocardial. The actinrelated protein 23 arp23 complex is the key component of these networks by virtue of its ability to initiate actin filament branches daughter filaments at an angle on the sides of preexisting mother filaments mullins et. Equilibrium thermodynamic analysis is frequently applied to.
Only full length and correctly folded actin can form a high affinity complex with dnase i. The higheraffinity complexes of actings1 and actindnase i were prepared by mixing at a molar ratio of 1. These molecules are visualized, downloaded, and analyzed by users who range from students. The rcsb pdb also provides a variety of tools and resources. The lowresolution camera was driven by the pmis software photometrics, which allows for macro programming, and the high resolution one by metamorph software universal imaging, west chester, pa with the use of predesigned algorithms for image acquisition and particle tracking. Actin may contribute to dna fragmentation following. Actins are highly conserved proteins that are involved in cell motility, structure and integrity. It has been shown that an atpase domain of actin shares similarity with atpase domains of hexokinase and hsp70 proteins pubmed. Bovine pancreatic dnase i shares 78% identity and super. Actin is a globular, roughly 42 kda protein found in literally all eukaryotic cells, where it may be present at concentrations of over 100. The structural basis of actin filament branching by the. Wiskottaldrich syndrome protein wasphomology domain 2 wh2 is a small and widespread actinbinding motif. In vitro dnase i is known to interact strongly with gactin in a 1.
We also assessed the fraction of folded to unfolded actin by measuring the fraction of folded actin monomer. The distribution of cofilin and dnase i in vivo cell. Expression of recombinant gfpactin fusion protein in the. Therefore, the changes in lightscattering intensity on subsequent titration of actin tm complex with s1 measure s1 binding to both actin tm and to free actin, which is reflected in a further increase of the intensity of scattered light at s1 concentrations exceeding the concentration of the actin tm complex. A common architecture, found in spire, cobl, vopl, and vopf, consists of tandem w domains that tie together three to four actin monomers to form a. D denotes partially denatured actin, which has lost dnase i binding capacity but retains approximately 70% of its ahelical structure. Mechanism of actin nterminal acetylation science advances. Here we describe the crystal structures of complexes of acti. The ability of actin to inhibit dnasei activity reflects the number of available dnasei binding sites in a sample of actin. Dnase iactin interaction the affinity of the actindnase i interaction was determined from doublereciprocal plots of the dependence of dnase i inhibition on actin concentration using the dnase i inhibition assay 17. The nterminal peptide comprised of residues 1207 of actin inhibits dnase i, while the tryptic fragments cleavage sites between residues 62 and 63 or between 68 and 69 fail to. Efficient binding of the chimera to the dnase i indicated nativity of the actin 5c fusion in vitro. The initiation of actin polymerization in cells requires actin filament nucleators.
Actin is a highly abundant intracellular protein present in all eukaryotic cells and has a pivotal role in muscle contraction as well as in cell movements. The dbp actin complex role of dbp in the actinscavenger system. Fluorescent dnase i has also been used as a model system to study the interactions of nucleotides, cations and cytochalasin d with monomeric. We offer highly purified actin from rabbit muscle, as well as fluorescent actin conjugates labeled with four of our brightest and most photostable dyes. Dnase i complex, suggesting that the nucleotide binding site in the actin. Dnase i complex adopts a conformation similar to that found in factin. Aug 19, 2008 in the profilinactin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4. The actin promoter deletions were obtained by cutting with xhi at position 272 and subsequently digesting with nuclease ba1. Schutt2 and roger karlsson1 1department of cell biology, the wennergren institute, stockholm university, sweden.
The structure of the dbpactin complex reveals that gactin binds in one of the dbp grooves, mainly formed by helix 10 of domain i, helix 6 of domain ii, and helix 3 of domain iii, allowing dbp and actin to fit as two pieces of a jigsaw puzzle. Cd spectra of the actin dnase i complex in the far and near insert uv region. The actinrelated protein 23 arp23 complex is the key component of these networks by virtue of its ability to initiate actin filament branches daughter filaments at an angle on the sides of preexisting mother. Actin alpha 1 which is expressed in skeletal muscle is one of six different actin isoforms which have been identified. Actin is the most abundant cytoskeletal protein in eukaryotic cells and forms a double. The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease i both in the atp and adp forms have been determined byo xray analysis at an effective resolution of 2. We used this feature of dnase i to identify phage binders that are captured by actin alone, but not the dnase i. The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease i both in the atp and adp forms have been determined by xray analysis at an effective resolution of 2. Engineering actin resistant human dnase i for treatment of cystic fibrosis article pdf available in proceedings of the national academy of sciences 9316.
Subsequently, actin structures with certain other abps were determined. Modulation of actin structure and function by phosphorylation. Users can perform simple and advanced searches based on annotations relating to sequence, structure and function. Actin is a 43 kda protein that is very highly conserved between species. The influence of cytochalasins on actin structure in monocytes has been quantitated by flow cytometry using texas red dnase i and bodipy fl phallacidin to stain the g actin and f actin pools, respectively.
The actinprofilin complex was prepared by mixing actin and profilin1 at a 1. With the exception of formins, known filament nucleators use the wiskottaldrich syndrome protein wasp homology 2 wh2 or w domain for interaction with actin. Cd spectra of the actindnase i complex in the far and near insert uv region. Crystal structure of actindnase i complex pdb accession number, 1atn dnase i purple interacts with loop on subdomain 2 of actin grey 15. The majority of the isotype heterogeneity is located in the aminoterminal 30 residues. Is it a globular or an intrinsically disordered protein. By immobilizing dnase i on sepharose beads, folded actin can be pulled down from solution and quantified by sdspage. Although much is now known about the clinical aspects of myopathies resulting from over 60 different acta1 mutations, we have very little evidence for how mutations alter the behavior of the actin. A tangle of crosslinked actin filaments fills the cytoplasm of animal, plant and fungal cells, forming a cytoskeleton that gives the cell shape and form and provides a scaffold for organization. Actin polymerization is essential for pollen tube growth. Dnase i is known to bind to the pointed end of g actin monomers but not well to f actin filaments. The binding of dnase i to actin is shown to be affected by antibodies specific to a central region in actin sequence 168226. The underside of actin in this figure contains the binding site of a number of abps including cofilin.
Structural basis for actin assembly, activation of atp. Here, we present the cryoelectron microscopic cryoem structure of filamentous actin factin in the presence of phosphate, with the visualization of some. Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. D kapp where n is native actin, and kapp is a temperaturedependent, apparent firstorder rate constant. Dnase i is known to bind to the pointed end of gactin monomers but not well to factin filaments. Crystal structure of actin dnase i complex pdb accession number, 1atn dnase i purple interacts with loop on subdomain 2 of actin grey 15. The surface area buried at the interface of this complex is as large as 3600 a 2. Crystal structure of smooth muscle g actin dnase i. A common architecture, found in spire, cobl, vopl, and vopf, consists of tandem w domains that tie. Threedimensional structure of bovine pancreatic dnase i at 2. The complex ultrastructure of cellstheir shape and internal structureand the many motions of cells are largely supported by filaments of actin. The gelsolin binding of actin, identified in the panactin coprecipitation, was 3.
The dnasei binding loop of actin may play a role in the regulation of actinmyosin interaction by tropomyosin. Dnase i is shown in gray with the site of fitc labeling indicated. The underside of actin in this figure contains the. Mutational analysis of arginine 177 in the nucleotide. Pathway of actin folding directed by the eukaryotic. Xray scattering study of actin polymerization nuclei. The dnase i binding loop residues 3852, the hydrophobic plug residues 262274, and the cterminus region are among the structural elements of monomeric g actin that were proposed to form the intermonomer interface in factin. Evidence for an factin like conformation in the actin. Crystal structure of actindnase i complex pdb accession number, 1atn dnase i purple interacts with loop on subdomain 2 of actin grey. Atp hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Fulllength gelsolin can bind with actin and deoxyribonuclease dnase i, a key enzyme responsible for dna degradation in apoptosis,14 in a noncompetitive manner to form a gelsolin actin dnase i ternary complex. It is known that one actin molecule tightly binds one atp or adp, and that transition from the monomeric state g.
Dnase i is a nuclease that cleaves dna preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5phosphateterminated polynucleotides with a free hydroxyl group on position 3, on average producing tetranucleotides. Generally, it is considered that unfolded actin aggregates do not bind dnase i and therefore, dnase i inhibition is often used to infer the proportion of folded and unfolded actin within a sample 20. This result is consistent with the hypothesis that the two major domains of actin on either side of the cleft are able to flex or move relative. As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. Actin mutations in hypertrophic and dilated cardiomyopathy. The structural basis of actin filament branching by the arp2. Cd spectroscopy proved to be a convenient technique for studying the interactions between ac tin and actinbinding proteins in solution. In the crystal complexes of gelsolinactin and profilinactin, the dnase i. The authors have investigated various structural and interaction properties of maleimidobenzoylgactin mbsactin, a new, internally crosslinked gactin derivative that does not exhibit, at moderate protein concentration, the saltand myosin subfragment 1 s1induced polymerizations of gactin and reacts reversibly and covalently in solution with s1 at or near. Cd spectroscopy proved to be a convenient technique for studying the interactions between ac tin and actin binding proteins in solution. The site on subdomain 4 is less extensive 156 a 2, but important, as it includes a salt bridge dnase i his44, actin glu207 and a hydrogen bond dnase i gln, actin thr203.
By monitoring the thermophoretic behavior of atto 488labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases. It is also one of the most highly conserved proteins, differing by no more than 20% in species as diverse as algae and humans. Deoxyribonuclease i usually called dnase i, is an endonuclease coded by the human gene dnase1. The dnasei binding loop of actin may play a role in the. Actin, alpha skeletal muscle is a protein that in humans is encoded by the acta1 gene. Structural basis for the slow dynamics of the actin. The greenfluorescent alexa fluor 488 actin conjugate has. The distribution of cofilin and dnase i in vivo cell research. This complex, when present intact, inhibits nuclear translocation and enzymatic activity of dnase i in vivo.
Refined structure and solvent network of chicken gizzard gactin dnase 1 complex at 1. Refined structure and solvent network of chicken gizzard g actin dnase 1 complex at 1. Previous observations suggest that actin filaments turn over rapidly within the apical region of the pollen tube. Fulllength gelsolin can bind with actin and deoxyribonuclease dnase i, a key enzyme responsible for dna degradation in apoptosis,14 in a noncompetitive manner to form a gelsolinactindnase i ternary complex.
Based on the knowledge that actin is the natural inhibitor of dnase i, 15 we next examined the interaction of gelsolin actin dnase i in the actin complex. The gelsolin binding of actin, identified in the pan actin coprecipitation, was 3. The dnase i binding loop residues 3852, the hydrophobic plug residues 262274, and the cterminus region are among the structural elements of monomeric g actin that were proposed to form the intermonomer interface in f actin. Salmonella enterica serovar typhimurium invades host macrophages and induces a unique caspase. It is the monomeric subunit of microfilaments, one of the three major components of the cytoskeleton, and of thin. As a result, the nucleotide cleft becomes moderately more open in the profilinactin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin. After cell destruction, purification of gfpactin 5c was performed by dnase isepharose. The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease i both in the atp and adp forms have been. Thermal unfolding of gactin monitored with the dnase i. Mutational analysis of arginine 177 in the nucleotide binding. Actin also has an essential function in maintaining and controlling cell shape and architecture. Dnase i complex adopts a conformation similar to that found in f actin.
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