Biomedical subjects

Vitold E Galkin

Publications and source records attributed to Vitold E Galkin.

18 recordsLinked to original sources

PubMed2007

Mapping the interaction of cofilin with subdomain 2 on actin.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.

Actin Depolymerizing Factors↗

PubMed2006

The CH-domain of calponin does not determine the modes of calponin binding to F-actin.

Many actin-binding proteins have been observed to have a modular architecture. One of the most abundant modules is the calponin-homology (CH) domain, found as tandem repeats in proteins that cross-link actin filaments (such as fimbrin, spectrin and alpha-actinin) or link the actin cytoskeleton to intermediate filaments (such as plectin). In proteins such as the eponymous calponin, IQGAP1, and Scp1, a single CH-domain exists, but there has been some controversy over whether this domain binds to actin filaments. A previous three-dimensional reconstruction of the calponin-F-actin complex has led to the conclusion that the visualized portion of calponin bound to actin belongs to its amino-terminal homology (CH) domain. We show, using a calponin fragment lacking the CH-domain, that this domain is not bound to F-actin, and cannot be positioning calponin on F-actin as hypothesized. Further, using classification methods, we show a multiplicity in cooperative modes of binding of calponin to F-actin, similar to what has been observed for other actin-binding proteins such as tropomyosin and cofilin. Our results suggest that the form and function of the structurally conserved CH-domain found in many other actin-binding proteins have diverged. This has broad implications for inferring function from the presence of structurally conserved domains.

Actins↗

PubMed2006

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity.

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.

Adenosine Triphosphatases↗

PubMed2005

Xin-repeats and nebulin-like repeats bind to F-actin in a similar manner.

Xin and nebulette are striated muscle-specific actin-binding proteins that both contain multiple actin-binding repeats. The nature of these repeats is different: nebulette has nebulin-like repeats, while Xin contains its own unique repeats. However, the suggestion was made from biochemical data that the Xin-repeats may bind to multiple sites on the actin molecule as was found for nebulin. We have used electron microscopy and the iterative helical real space reconstruction to visualize complexes of F-actin with Xin fragments containing either three or six Xin-repeats, and with the CN5-nebulette fragment, containing five nebulin-like repeats. Our results indicate that Xin and nebulette fragments bind to F-actin in a similar manner and in two distinct modes: in one mode actin subdomain 1 is bound, while in the second mode the binding bridges between a different site on actin subdomains 1/2 of one protomer and subdomains 3/4 of an adjacent actin protomer. Taken together with published data about nebulin, tropomyosin and ADF/cofilin, our results suggest that the ability to bind in multiple modes to the actin protomer is a general property of many actin-binding proteins.

Actins↗

PubMed2005

BRCA2 BRC motifs bind RAD51-DNA filaments.

Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.

BRCA2 Protein↗

PubMed2005

Refining the structure of the Halobacterium salinarum flagellar filament using the iterative helical real space reconstruction method: insights into polymorphism.

The eubacterial flagellar filament is an external, self-assembling, helical polymer approximately 220 A in diameter constructed from a highly conserved monomer, flagellin, which polymerizes externally at the distal end. The archaeal filament is only approximately 100 A in diameter, assembles at the proximal end and is constructed from different, glycosylated flagellins. Although the phenomenology of swimming is similar to that of eubacteria, the symmetry of the archebacterial filament is entirely different. Here, we extend our previous study on the flagellar coiled filament structure of strain R1M1 of Halobacterium salinarum. We use strain M175 of H.salinarum, which forms poly-flagellar bundles at high yield which, under conditions of relatively low ionic-strength (0.8 M versus 5 M) and low pH ( approximately 2.5 versus approximately 6.8), form straight filaments. We demonstrated previously that a single-particle approach to helical reconstruction has many advantages over conventional Fourier-Bessel methods when dealing with variable helical symmetry and heterogeneity. We show here that when this method is applied to the ordered helical structure of the archebacterial uncoiled flagellar filament, significant extensions in resolution can be obtained readily when compared to applying traditional helical techniques. The filament population can be separated into classes of different morphologies, which may represent polymorphic states. Using cryo-negatively stained images, a resolution of approximately 10-15 A has been achieved. Single alpha-helices can be fit into the reconstruction, supporting the proposed similarity of the structure to that of type IV bacterial pili.

Biopolymers↗

PubMed2004

The Arg non-receptor tyrosine kinase modifies F-actin structure.

The Arg (Abl-related gene) protein belongs to the Abl family of non-receptor tyrosine kinases that regulate cell motility and morphogenesis. It contains two actin-binding domains, one containing the talin-like I/LWEQ motif, and a C-terminal calponin homology (CH) domain. We used electron microscopy and single particle image analysis to reconstruct complexes of F-actin with full-length Arg, and fragments lacking either the I/LWEQ or CH domains. The Arg CH domain binds to actin's subdomain-1 (SD1) and induces a tilt of actin protomers. The I/LWEQ domain binds to either SD1 or SD4, closing the nucleotide binding cleft of actin. Although Arg can use either its CH or ILWEQ domains to bind an actin filament, both domains within Arg cannot bind simultaneously to adjacent protomers in the filament, consistent with its F-actin-bundling activity. The conformational changes in the filament introduced by Arg can explain the cooperative binding of Arg to F-actin and might prevent other actin binding proteins from binding to actin filaments.

Actins↗

PubMed2004

Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization.

Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly being polymerized and depolymerized within cells as part of cellular motility, tissue formation and repair, and embryonic development. Many proteins exist that bind to monomeric or filamentous (F) forms of actin to regulate the polymerization state. It has become increasingly apparent that the ability of different proteins to bind to and regulate actin filament dynamics depends on the ability of the filament to exist in altered conformations. Yet, little is known about how these conformational changes occur at the molecular level. We have destabilized F-actin filaments by forming a disulfide that locks the "hydrophobic plug" to the body of the actin subunit or by altering the C terminus of actin with a tetramethylrhodamine label. We also examined F-actin filaments at short times after the initiation of polymerization. In all three cases, a substantial fraction of protomers can be found in a "tilted" state that also is induced by actin depolymerizing factor/cofilin proteins. These observations suggest that F-actin filaments are annealed over time into a stable filament and that actin-depolymerizing proteins can effect a time reversal of polymerization.

Actin Cytoskeleton↗

PubMed2003

ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments.

Proteins in the ADF/cofilin (AC) family are essential for rapid rearrangements of cellular actin structures. They have been shown to be active in both the severing and depolymerization of actin filaments in vitro, but the detailed mechanism of action is not known. Under in vitro conditions, subunits in the actin filament can treadmill; with the hydrolysis of ATP driving the addition of subunits at one end of the filament and loss of subunits from the opposite end. We have used electron microscopy and image analysis to show that AC molecules effectively disrupt one of the longitudinal contacts between protomers within one helical strand of F-actin. We show that in the absence of any AC proteins, this same longitudinal contact between actin protomers is disrupted at the depolymerizing (pointed) end of actin filaments but is prominent at the polymerizing (barbed) end. We suggest that AC proteins use an intrinsic mechanism of F-actin's internal instability to depolymerize/sever actin filaments in the cell.

Actin Depolymerizing Factors↗

PubMed2003

Complexes of RecA with LexA and RecX differentiate between active and inactive RecA nucleoprotein filaments.

The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.

Bacterial Proteins↗

PubMed2003

Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms.

Like many bacterial pathogens, Salmonella spp. use a type III secretion system to inject virulence proteins into host cells. The Salmonella invasion protein A (SipA) binds host actin, enhances its polymerization near adherent extracellular bacteria, and contributes to cytoskeletal rearrangements that internalize the pathogen. By combining x-ray crystallography of SipA with electron microscopy and image analysis of SipA-actin filaments, we show that SipA functions as a "molecular staple," in which a globular domain and two nonglobular "arms" mechanically stabilize the filament by tethering actin subunits in opposing strands. Deletion analysis of the tethering arms provides strong support for this model.

Actin Cytoskeleton↗

PubMed2003

Do the utrophin tandem calponin homology domains bind F-actin in a compact or extended conformation?

Tandem calponin-homology (CH) domains play an important role in the actin-binding function of many spectrin superfamily proteins. Crystal structures from several of these proteins have suggested a flexibility between these domains, and the manner in which these domains bind to F-actin has been the subject of some controversy. A recent paper has used electron microscopy and three-dimensional reconstruction to examine the complex of the utrophin tandem CH domain with F-actin. In contrast to our previously published study, a closed conformation of the two calponin-homology domains was suggested in the new work. We show here that the new results can be explained by incomplete binding of utrophin to actin, heterogeneity in the mode of binding, and angular disorder in F-actin. We conclude that helical averaging applied to disordered filaments is responsible for their results, and that approaches designed to separate out homogeneous subsets within such filamentous complexes offer many advantages.

Actins↗

PubMed2003

The location of ubiquitin in Lethocerus arthrin.

Arthrin is a ubiquitinated actin that is present in flight muscles of some insects. In addition, it has been found in the malaria parasite Plasmodium falciparum. The role of this monoubiquitylation is not clear, and it does not appear to be associated with proteolytic degradation. The stoichiometry of arthrin to actin in Lethocerus indirect flight muscle, 1:6, suggests that there would be one arthrin molecule for each Tm-Tn (tropomyosin-troponin) complex. The appearance of arthrin after tropomyosin and troponin in Drosophila development is consistent with the Tm-Tn complex determining which actin subunit is targeted for conjugation with ubiquitin. We have used a new approach of three-dimensional reconstruction of helical filaments, the iterative helical real space reconstruction method, to extract segments of homogeneous arthrin out of long filaments where the conformation of the ubiquitin is more heterogeneous. Surprisingly, the location of the ubiquitin is on the face of actin subdomain 1, opposite to where tropomyosin binds in the "off" state, suggesting that there could not be a direct interaction between the ubiquitin and the tropomyosin. It is possible that the troponin complex in the "on" state that is bound to one actin strand makes an unfavorable contact with a ubiquitin molecule attached to the opposite actin strand. This might be the basis for a destabilization of the on state at rest length. Lys118 is the most likely residue to which the ubiquitin is conjugated, based upon fitting atomic structures of actin and ubiquitin into the reconstruction.

Actins↗

PubMed2002

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.

Actins↗

PubMed2002

A new internal mode in F-actin helps explain the remarkable evolutionary conservation of actin's sequence and structure.

Actin is one of the most highly conserved eukaryotic proteins. There are no amino acid changes between the chicken and human skeletal muscle isoforms, and the most dissimilar actins still share more than 85% sequence identity [1]. We suggest that large discrete internal modes of freedom within the actin filament may account for a significant component of this conservation, since each subunit must make multiple specific interactions with neighboring subunits. In support of this, we find that the same state of tilt of the actin subunit exists in both yeast and vertebrate striated muscle actin, and that in both the two domains undergo a "propeller rotation." A similar movement of domains has also been seen in hexokinase, Hsc70, and Arp2/3, all structural homologs of actin, suggesting that such an interdomain hinge motion is common to proteins in this superfamily. Subunit-subunit interactions within the actin filament involve sequence insertions that are not present in MreB, a bacterial homolog of actin. Remarkably, we find that in the tilted state actin subunits make new contacts with neighboring subunits that also involve these inserts, suggesting a key role for these elements in F-actin polymorphism.

Actin Depolymerizing Factors↗

PubMed2002

Each actin subunit has three nebulin binding sites: implications for steric blocking.

Nebulin is a giant protein that spans most of the muscle thin filament. Mutations in nebulin result in myopathies and dystrophies. Nebulin contains approximately 200 copies of approximately 35 residue modules, each believed to contain an actin binding site, organized into seven-module superrepeats. The strong correlation between the number of nebulin modules and the length of skeletal muscle thin filaments in different species suggests that nebulin determines thin filament length. Little information exists about the interactions between intact nebulin and F-actin. More insight has come from working with fragments of nebulin, containing from one to hundreds of actin binding modules. However, the observed stoichiometry of binding between these fragments and actin has ranged from 0.4 to 13 modules per actin subunit. We have used electron microscopy and a novel method of helical image analysis to characterize complexes of F-actin with a nebulin fragment. The fragment binds as an extended structure spanning three actin subunits and binding to different sites on each actin. Muscle regulation involves tropomyosin movement on the surface of actin, with binding in three states. Our results suggest the intriguing possibility that intact nebulin may also be able to occupy three different sites on F-actin.

Actins↗

PubMed2002

The bacterial protein SipA polymerizes G-actin and mimics muscle nebulin.

SipA is a Salmonella protein delivered into host cells to promote efficient bacterial entry, which is essential for pathogenicity. SipA exerts its function by binding F-actin, resulting in the stabilization of F-actin and the stimulation of the bundling activity of fimbrin. Here we show that under low salt conditions where spontaneous nucleation and polymerization of actin do not occur, SipA induces extensive polymerization. We have used electron microscopy and a method for helical image analysis to visualize the complex of actin with the actin-binding fragment of SipA. The SipA fragment binds to actin as a tubular molecule extending approximately 95 A. The main sites of SipA binding on actin involve sequence insertions that are not present in the bacterial homolog of actin, MreB, suggesting a mechanism for preventing SipA from interacting with bacterial MreB filaments. Remarkably, the pattern of SipA binding, which connects subunits on opposite actin strands and explains the stabilization of F-actin, is similar to that shown for a fragment of the giant muscle protein nebulin. We suggest that SipA is a bacterial structural mimic of muscle nebulin and nebulin-like proteins in non-muscle cells that are involved in the regulation of the actin-based cytoskeleton.

Actins↗

PubMedDate not supplied

Issues of resolution and polymorphism in single-particle reconstruction.

Three-dimensional reconstruction from electron microscopic (EM) images of isolated macromolecular complexes is being employed by many laboratories. This approach is extremely powerful and continues to improve in resolution. In the absence of stereochemical constraints that can be used to assess the quality of a reconstruction, as exist in X-ray crystallography, several other measures have typically been used. A very useful assessment of quality can be made in the comparison between the projections of the three-dimensional reconstruction and averages generated from classes of images. The main quantitative measure has been that of resolution statistics, typically based upon Fourier shell correlations. We show, using only simulated noise for images, that impressive resolution statistics are generated that can even extend the apparent resolution of the starting model. When truly independent reconstructions are generated starting from different initial models, however, such artefacts are not possible. We also show, using real images of DnaB rings, that in the presence of polymorphism artefactual reconstructions can be generated whose projections match class averages. These averages, however, are themselves artefactual as they involve heterogeneous images. The issues presented here need to be considered when single-particle EM reconstructions are evaluated.

Bacterial Proteins↗