Multiple Conformations of F-actin

Actin works within eukaryotic cells to facilitate a variety of cellular processes, which are driven by the assembly of G-actin (monomeric form) into F-actin (fibrous form), and the disassembly of F-actin into G-actin. F-actin adopts multiple conformations, which are specified by interactions with various actin-binding proteins. Knowledge of the multiple conformations of actin is the key for understanding its cellular functions. Recently, we published a refined model for F-actin. In this review, based on this model, we discuss the origin, mechanism, and possible physiological significance of the multiple conformations of F-actin

Capping protein binding to actin in yeast: biochemical mechanism and physiological relevance

The mechanism by which capping protein (CP) binds barbed ends of actin filaments is not understood, and the physiological significance of CP binding to actin is not defined. The CP crystal structure suggests that the COOH-terminal regions of the CP α and β subunits bind to the barbed end. Using purified recombinant mutant yeast CP, we tested this model. CP lacking both COOH-terminal regions did not bind actin. The α COOH-terminal region was more important than that of β. The significance of CP's actin-binding activity in vivo was tested by determining how well CP actin-binding mutants rescued null mutant phenotypes. Rescue correlated well with capping activity, as did localization of CP to actin patches, indicating that capping is a physiological function for CP. Actin filaments of patches appear to be nucleated first, then capped with CP. The binding constants of yeast CP for actin suggest that actin capping in yeast is more dynamic than in vertebrates

Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells

AbstractA new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P43(1)212 with cell constants of a = b = 105.6 Å, c = 506.9 Å. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of α-helical coiled-coil. Thus in the region of 10-5.5 Å resolution along the c∗-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 Å along the c∗-axis, whereas along the a∗–b∗-plane reflections were limited to 6.6 Å. Data analysis is under way on a data set from a PtCl4 derivative

Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG–O3B bond cleavage; 3) four concomitant events: W1–PO3− formation, OH− and proton cleavage, nucleophilic attack by the OH− against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes

Two Distinct Mechanisms for Actin Capping Protein Regulation—Steric and Allosteric Inhibition

A crystallographic study reveals the structural basis for regulation by two different inhibitors of the actin capping protein, a critical factor controlling actin-driven cell motility

Complete coding sequences of cDNAs of four variants of rabbit skeletal muscle troponin T

Four variants of troponin T (TnT) cDNAs have been isolated and sequenced. These cDNAs have been derived from rabbit skeletal muscle, the most widely studied source of troponin, of a 11-day-old animal. One variant (TnT-1) contains the complete coding sequence, while in three variants the coding sequences are truncated at the 5′ termini. The previously published amino acid sequence differs from the present cDNA-derived sequences at three locations. At least two, possibly all, of them are probably accounted for by errors in peptide sequencing. The present results are consistent with the two types of alternative splicing of TnT genes, both being first reported on the rat gene. (1) Highly variable sequences in the amino-terminal region are accounted for by the alternative splicing of exons 4–8 in an interchangeable but not mutually exclusive manner. (2) In the carboxyl-terminal region, the alternative splicing of two exons 17 (β-type) or 16 (α-type) in mutually exclusive manner is consistent with the difference between all the four cDNAs, which express exon 17, and the previously published peptide sequence (derived from the adult muscle) in which exon 16 is present. This variation also corresponds to the finding in chicken skeletal muscle that the choice of exon 16 or 17 may be dependent on developmental stages. Finally, a sequence is observed corresponding to an extra exon or exons between exons 5 and 6. This sequence is shorter than that of the chicken skeletal muscle gene and is not detected in the rat skeletal muscle gene

Calcium Ions and the Structure of Muscle Actin Filament An X-ray Diffraction Study

In order to investigate the effects of $Ca^{2+}$-binding to troponin on the conformation of the muscle thin filament (consisting of actin, tropomyosin and troponin) in the absence of actomyosin interaction, two series of X-ray diffraction experiments were undertaken. Firstly, the small angle X-ray scattering from filaments in solution indicate the tropomyosin strands are centred at about 3·5 nm from the filament axis and this distance is calcium independent. Secondly, X-ray fibre diffraction, patterns from the filaments orientated in glass capillaries were studied. The X-ray intensity of the 2nd actin layer-line increased in a highly co-operative manner at a concentration of free calcium ions [Ca$^{2+}$] of 10-6·8 M, which is the range in which muscle contraction is physiologically regulated. However, this intensity increase accounted for some 30% of the total increase observed in diffraction patterns from muscle on activation, suggesting that the Ca$^{2+}$-binding alters the state of the thin filament, which then undergoes further changes upon interaction with myosin

Crystal structure of CapZ: structural basis for actin filament barbed end capping

Capping protein, a heterodimeric protein composed of α and β subunits, is a key cellular component regulating actin filament assembly and organization. It binds to the barbed ends of the filaments and works as a ‘cap’ by preventing the addition and loss of actin monomers at the end. Here we describe the crystal structure of the chicken sarcomeric capping protein CapZ at 2.1 Å resolution. The structure shows a striking resemblance between the α and β subunits, so that the entire molecule has a pseudo 2-fold rotational symmetry. CapZ has a pair of mobile extensions for actin binding, one of which also provides concomitant binding to another protein for the actin filament targeting. The mobile extensions probably form flexible links to the end of the actin filament with a pseudo 2(1) helical symmetry, enabling the docking of the two in a symmetry mismatch

Crystal structure of the C-terminal half of tropomodulin and structural basis of actin filament pointed-end capping.

Tropomodulin is the unique pointed-end capping protein of the actin-tropomyosin filament. By blocking elongation and depolymerization, tropomodulin regulates the architecture and the dynamics of the filament. Here we report the crystal structure at 1.45-A resolution of the C-terminal half of tropomodulin (C20), the actin-binding moiety of tropomodulin. C20 is a leucine-rich repeat domain, and this is the first actin-associated protein with a leucine-rich repeat. Binding assays suggested that C20 also interacts with the N-terminal fragment, M1-M2-M3, of nebulin. Based on the crystal structure, we propose a model for C20 docking to the actin subunit at the pointed end. Although speculative, the model is consistent with the idea that a tropomodulin molecule competes with an actin subunit for a pointed end. The model also suggests that interactions with tropomyosin, actin, and nebulin are all possible sources of influences on the dynamic properties of pointed-end capping by tropomodulin

Reconstitution of Rabbit Skeletal Muscle Troponin from the Recombinant Subunits All Expressed in and Purified from E. coli

Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are β-TnT(N'–208), TnI(C64A, C133S), and the wild type TnC. β–TnT(N'–208) is a 25 kDa fragment of y9-troponin T, which consists of 208 amino acids and lacks 58 residues in the N–terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys–64 and Cys–133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-Sl ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials