Structural basis for cofilin binding and actin filament disassembly.

Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin-actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin-actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments.

Calcium-dependent interaction sites of tropomyosin on reconstituted muscle thin filaments with bound Myosin heads as studied by site-directed spin-labeling.

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca(2+) ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca(2+). These findings suggest that even in the myosin-bound (open) state, Ca(2+) may regulate actomyosin contractile properties via Tm.

A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament.

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin-tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146-174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor-acceptor pairs, FRET efficiencies were determined with and without Ca(2+). Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146-174 on F-actin to calculate the FRET efficiency for each donor-acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41-69, 83-111, 216-244, and 252-279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217-236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca(2+) than with it. Ca(2+)-induced changes on the actin-Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.

Interaction sites of tropomyosin in muscle thin filament as identified by site-directed spin-labeling.

To identify interaction sites we measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, and 279. Upon the addition of F-actin, the mobility of all the spin labels, especially at position 13, 271, or 279, of Tm was inhibited significantly. Slow spin-label motion at the C-terminus (at the 230th and 271st residues) was observed upon addition of troponin. The binding of myosin-head S1 fragments without troponin immobilized Tm residues at 146, 160, 190, 209, 230, 271, and 279, suggesting that these residues are involved in a direct interaction between Tm and actin in its open state. As immobilization occurred at substoichiometric amounts of S1 binding to actin (a 1:7 molar ratio), the structural changes induced by S1 binding to one actin subunit must have propagated and influenced interaction sites over seven actin subunits.

Electron microscopic visualization of the filament binding mode of actin-binding proteins.

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.

Fluorescence resonance energy transfer between residues on troponin and tropomyosin in the reconstituted thin filament: modeling the troponin-tropomyosin complex.

Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle alpha-Tm, TnI and the beta-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca(2+)-induced conformational change of the Tm-Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn-Tm complex with and without bound Ca(2+). In the best-fit models, the Tn core domain is located on residues 160-200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is approximately 75 degrees and approximately 85 degrees with and without bound Ca(2+), respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca(2+), and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by approximately 20 degrees upon Ca(2+) binding.

The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments.

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.