Margaret S. VanLoock

ATP-mediated conformational changes in the RecA filament.

The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecAs activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.

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.

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-actins internal instability to depolymerize/sever actin filaments in the cell.

The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings.

Mini‐chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C‐terminal helicase domain that can be fit with crystal structures of ring helicases and an N‐terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.

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.

Flexibility of the rings: Structural asymmetry in the DnaB hexameric helicase

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.

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.

How does ATP hydrolysis control actin's associations?

Polymers of actin (F-actin) form an integral part of the structural framework that supports the plasma membrane of our cells while providing a platform for signaling and metabolic proteins. Most subunits in an actin filament hydrolyze a single molecule of ATP to ADP over the F-actins lifetime. This hydrolysis is the critical timekeeper of F-actin longevity that informs a host of accessory proteins about the state of the filament (1). Here, we discuss the structural changes within each subunit of F-actin that are induced by the nucleotide hydrolysis.

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 ∼95 Å. 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.

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes.

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.