Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.

Actin filaments, stereocilia, and hair cells of the bird cochlea. II. Packing of actin filaments in the stereocilia and in the cuticular plate and what happens to the organization when the stereocilia are bent.

A comparison of hair cells from different parts of the cochlea reveals the same organization of actin filaments; the elements that vary are the length and number of the filaments. Thin sections of stereocilia reveal that the actin filaments are hexagonally packed and from diffraction patterns of these sections we found that the actin filaments are aligned such that the crossover points of adjacent actin filaments are in register. As a result, the cross-bridges that connect adjacent actin filaments are easily seen in longitudinal sections. The cross-bridges appear as regularly spaced bands that are perpendicular to the axis of the stereocilium. Particularly interesting is that, unlike what one might predict, when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. In the images of bent bundles, the bands of cross-bridges are then tilted off perpendicular to the stereocilium axis. When the stereocilium is bent at its base, all cross-bridges in the stereocilium are affected. Thus, resistance to bending or displacement must be property of the number of bridges present, which in turn is a function of the number of actin filaments present and their respective lengths. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. Perhaps its physiological vulnerability may result from changes of stereociliary structure.

The location of DNA in RecA-DNA helical filaments.

The helical filament that the RecA protein of Escherichia coli forms around DNA is the active apparatus in protein-catalyzed homologous genetic recombination. The actual position of DNA within this complex has been unknown. Image analysis has been performed on electron micrographs of filaments of RecA on double-stranded DNA and on single-stranded DNA to visualize a difference that is consistent with one strand of the double-stranded DNA. This localization of the DNA gives additional information about the unusual structure of DNA in the complex with RecA protein.

Similarity of the yeast RAD51 filament to the bacterial RecA filament.

The RAD51 protein functions in the processes of DNA repair and in mitotic and meiotic genetic recombination in the yeast Saccharomyces cerevisiae. The protein has adenosine triphosphate-dependent DNA binding activities similar to those of the Escherichia coli RecA protein, and the two proteins have 30 percent sequence homology. RAD51 polymerized on double-stranded DNA to form a helical filament nearly identical in low-resolution, three-dimensional structure to that formed by RecA. Like RecA, RAD51 also appears to force DNA into a conformation of approximately a 5.1-angstrom rise per base pair and 18.6 base pairs per turn. As in other protein families, its structural conservation appears to be stronger than its sequence conservation. Both the structure of the protein polymer formed by RecA and the DNA conformation induced by RecA appear to be general properties of a class of recombination proteins found in prokaryotes as well as eukaryotes.

Actin filament structure. The ghost of ribbons past.

The high-resolution structures now available for monomeric actin, and for the actin-binding proteins profilin, gelsolin and myosin, have important implications for structural models of the actin filament.

Molecular evolution: actin's long lost relative found.

The bacterial protein MreB has been identified as a prokaryotic homolog of the eukaryotic cytoskeletal protein actin. While we still know little about MreB's function, the structural similarities and differences between MreB and actin provide more insight into the remarkable properties of actin.

Bacterial conjugation: running rings around DNA.

Helicases are active in many aspects of DNA replication, recombination, repair and transcription. An integral membrane bacterial protein assembly involved in the transfer of DNA between cells has been shown to resemble a ring helicase, suggesting that it hydrolyzes ATP to pump DNA through a central channel.

Tubulin family: kinship of key proteins across phylogenetic domains.

Atomic structures obtained by electron microscopy for tubulin, and by X-ray crystallography for bacterial FtsZ, show that the two proteins are highly homologous. The complementarity between such high-resolution studies and low-resolution reconstructions of microtubule complexes is clear, but controversy still abounds.

At least one of the protofilaments in flagellar microtubules is not composed of tubulin.

BACKGROUND: The core of the eukaryotic flagellum is the axoneme, a complex motile organelle composed of approximately 200 different polypeptides. The most prominent components of the axoneme are the central pair and nine outer doublet microtubules. Each doublet microtubule contains an A and a B tubule; these are composed, respectively, of 13 and 10-11 protofilaments, all of which are thought to be made of tubulin. The mechanisms that control the assembly of the doublet microtubules and establish the periodic spacings of associated proteins, such as dynein arms and radial spokes, are unknown. Tektins, a set of microtubule-associated proteins, are present in the axoneme as stable filaments that remain after the extraction of doublet microtubules; they are localized near to where the B tubule attaches to the A tubule and near to the binding sites for radial spokes, inner dynein arms and nexin links. Tektin filaments may contribute in an interesting way to the structural properties of axonemes. RESULTS: We have fractionated doublet microtubules from sea urchin sperm flagella into ribbons of stable protofilaments, which can be shown to originate from the A tubule. Using cryo-electron microscopy, conventional electron microscopy, scanning transmission electron microscopy, three-dimensional reconstruction and kinesin decoration, we have found that one protofilament in the ribbon is not composed of tubulin. This protofilament is an integral protofilament of the A tubule wall, has less mass per unit length than tubulin and does not bind kinesin. CONCLUSION: Contrary to what is generally assumed, at least one protofilament in the wall of the A tubule is not composed of tubulin. Our data suggest that this nontubulin protofilament is primarily composed of tektins, proteins that show some structural similarity to intermediate filament proteins. A 480 A axial periodicity within these ribbons, revealed by scanning transmission electron microscopy, can be related to the structure of tektin, and may determine the large-scale structure of the axoneme in terms of the binding of dynein, nexin and radial spokes to the doublet microtubule.

The human Rad52 protein exists as a heptameric ring.

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface.

ADF/cofilins are abundant actin binding proteins critical to the survival of eukaryotic cells. Most ADF/cofilins bind both G and F-actin, sever the filaments and accelerate their treadmilling. These effects are linked to rearrangements of interprotomer contacts, changes in the mean twist, and filament destabilization by ADF/cofilin. Paradoxically, it was reported that under certain in vitro and in vivo conditions cofilin may stabilize actin filaments and nucleate their formation. Here, we show that yeast cofilin and human muscle cofilin (cofilin-2) accelerate the nucleation and elongation of ADP-F-actin and stabilize such filaments. Moreover, cofilin rescues the polymerization of the assembly incompetent tethramethyl rhodamine (TMR)-actin and T203C/C374S yeast mutant actin. Filaments of cofilin-decorated TMR-actin and unlabeled actin are indistinguishable, as revealed by electron microscopy and three-dimensional reconstruction. Our data suggest that ADF/cofilins play an active role in establishing new interprotomer interfaces in F-actin that substitute for disrupted (as in TMR-actin and mutant actin) or weakened (as in ADP-actin) longitudinal contacts in filaments.

New insights into actin filament dynamics.

Great progress has been made in advancing an atomic-level model for F-actin. A growing body of data shows, however, that any picture of F-actin must take into account allosteric interactions within subunits, long-range cooperative effects that occur between subunits, and the fact that several conformations of the filament can exist.

Homomorphous hexameric helicases: tales from the ring cycle.

Low-resolution structures have now been determined for several hexameric ring proteins, members of a large superfamily that includes helicases and, probably, a range of DNA-binding motors. A common symmetry and mode of DNA-binding may emerge.

New angles on actin dynamics.

Actin is now realised to play a dynamic role in muscle contraction and many cellular motility events that occur when the motor domain of myosin uses the energy of ATP hydrolysis to move along the actin filament. Optical and electron microscopic studies have led to seemingly contradictory pictures of actin filament dynamics.

Does a stretched DNA structure dictate the helical geometry of RecA-like filaments?

Proteins in the RecA/Rad51/RadA/UvsX family form helical filaments on DNA in which the DNA is stretched and untwisted. A comparison of the average helical parameters of these filaments from five different proteins, obtained from archaea, eubacteria and eukaryotes, suggests that an intrinsic state of DNA may be responsible for the conservation of these particular filament forms across evolution. In this view, these proteins stabilize this existing state of DNA, rather than induce a novel conformation.

DNA conformation induced by the bacteriophage T4 UvsX protein appears identical to the conformation induced by the Escherichia coli RecA protein.

The study of homologous genetic recombination has been dominated by the RecA protein of Escherichia coli, which is involved in DNA recombination and repair, as well as phage induction, in vivo. The active form of the RecA protein is a helical filament formed on DNA in the presence of ATP, and within this filament, the DNA is extensively stretched to about 5.1 A rise per base-pair and untwisted to about 19 base-pairs per turn. The bacteriophage T4 UvsX protein is only weakly homologous to RecA, but it has very similar ATP-dependent DNA binding and strand-exchange activities. We can now show that the UvsX protein forms helical filaments that are very similar to those made by RecA, and induces the same extended DNA conformation within these filaments that is induced by RecA. This implies that the unusual conformation of DNA in the RecA filament may be a universal structure in homologous recombination.

A conformational change in the actin subunit can change the flexibility of the actin filament.

The mechanical properties of F-actin are very significant, given the central structural role played by actin filaments within muscle and the cytoskeleton. We have determined that actin can exist in a state that has a fourfold increase in flexibility over normal F-actin, and nucleotide. Three-dimensional reconstructions from electron micrographs suggest that this increased flexibility arises from a rotation of subdomain-2, the smallest subdomain, of the actin subunit. The modulation of actin's flexibility by Ca2+ and Mg2+ may have important physiological consequences within the cell. Further, since it has been shown that myosin-decorated actin filaments are more flexible than pure F-actin, it is possible that myosin induces this more flexible state in actin.

Angular disorder in actin: is it consistent with general principles of protein structure?

Harold Erickson has recently provided a useful analysis of helical structures having one class versus two classes of intersubunit bonds. His analysis is based upon an assumption that the subunits themselves are essentially unchanged upon bond formation (polymerization). He shows that such a structure having two classes of bonds (i.e. one in which each subunit interacts with four of its neighbors rather than two) can explain some of the features of actin. While he acknowledges that for actin there could be a conformational change and that, in principle, it could explain such features, he argues that the allowed magnitude of such a conformational change is inadequate. Since kinetics and thermodynamics cannot distinguish between the energy derived from the formation of a bond from that due to a conformational change, the question of whether the features of F-actin are derived from a conformational change or a system of two classes of bonds or both must be answered with high-resolution structural information. Recent studies by K. C. Holmes and others suggest that the second possibility might be closest to the truth. The heart of our disagreement is not whether Erickson's thermodynamic analysis is correct, given rigid subunits, but whether all protein polymers are characterized by rigid subunits with rigid intersubunit contacts. Erickson maintains that the observation of an angular disorder of 12 degrees per subunit within the actin filament conflicts with his formalism of rigid subunit interfaces and must therefore result from the erroneous interpretation of measurements. He presents an alternative model to explain the observations. His model, however, does not account for the observations and we will argue that, ultimately, like the resolution of the matter of the number of classes of bonds and the extent of their contact, the amount of angular disorder will require higher-resolution structural studies.

Structure of helical RecA-DNA complexes. Complexes formed in the presence of ATP-gamma-S or ATP.

Electron micrographs of RecA-DNA filaments, formed under several different conditions, have been analyzed and the filament images reconstructed in three dimensions. In the presence of ATP and a non-hydrolyzable ATP analog. ATP-gamma-S, the RecA protein forms with DNA a right-handed helical complex with a pitch of approximately 95 A. The most detailed view of the filament was obtained from analysis of RecA filaments on double-stranded DNA in the presence of ATP-gamma-S. There are approximately six subunits of RecA per turn of the helix, but both this number and the pitch are variable. From the examination of single filaments and filament-filament interactions, a picture of an extremely flexible protein structure emerges. The subunits of RecA protein are seen to be arranged in such a manner that the bound DNA must be partially exposed and able to come into contact with external DNA molecules. The RecA structure determined in the presence of ATP-gamma-S appears to be the same as the "pre-synaptic" state that occurs with ATP, in which there is recognition and pairing between homologous DNA molecules.

The LexA repressor binds within the deep helical groove of the activated RecA filament.

The RecA protein of Escherichia coli, as a result of DNA damage, catalyzes the cleavage of its own repressor, the LexA protein, and thereby initiates the SOS response. Using a non-cleavable LexA mutant, we have obtained a co-complex of both the RecA and LexA proteins on DNA. Mass analysis using scanning transmission electron microscopy suggests that the site size of the LexA repressor on RecA is two, which would be consistent with a nearest-neighbor exclusion model for binding. Three-dimensional reconstruction of electron micrographs of these filaments shows that the LexA protein is bound in the deep groove of the RecA filament, with two strong regions of contact that span adjacent RecA protomers within the filament. One contact is consistent with a proposed LexA binding site in the RecA crystal structure. The other contact maps onto a region that has been postulated to be a second DNA-binding site within RecA, which can explain the inhibition of RecA cleavage of LexA by excess DNA.