Gestalt-binding of tropomyosin on actin during thin filament activation

Abstract

Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

Figure 1

Control of tropomyosin position by troponin and Ca²⁺. Cross-sections of three superposed reconstructions showing the EM envelopes of reconstituted thin filaments (viewed from the pointed end of actin) consisting of troponin-free F-actin-tropomyosin (green), as well as troponin-decorated F-actin-tropomyosin maintained at low-Ca²⁺ (magenta) or treated with high-Ca²⁺ concentrations (yellow). Two azimuthally related actin subunits (Oda et al. 2009) (yellow, brown) were fitted within the reconstructions for reference, with acidic residues (red), basic residues (blue), and Pro333 (white, arrows) highlighted. Double-sided arrows indicate tropomyosin positions on one side of the filament. Note the good positional discrimination between high- and low-Ca²⁺ positions of tropomyosin on the inner and outer domains of actin for the troponin-regulated filaments, but the broader positioning of tropomyosin on the troponin-free filament. Also note, the position of actin residue Asp25 (asterisk), which is both a binding site for tropomyosin and a structural barrier restricting troponin-induced tropomyosin movement. Tropomyosin reconstructions were rendered with the program Chimera (Pettersen et al. 2004) based on data in Lehman et al. (2009) and Li et al. (2011).

Figure 2

Surface residues on the actin interface specify tropomyosin positioning. (a) An actin filament model showing residues defining the axial path over which tropomyosin runs on troponin-free actin (actin subdomain numbers marked, acidic (red) and basic (blue) amino acids that interact with tropomyosin are highlighted, Pro333 colored white (actin’s pointed end facing up)). (b) Enlargement of a single actin subunit viewed face-on and in (c) viewed end-on (from actin’s barbed end with only the top surface of the actin shown). Charged residues (Lys326, Lys328, Arg147, and Asp25) project from actin (Oda et al. 2009) and are poised to interact with tropomyosin (Li et al. 2011); in (c) the 326/328/147 residue cluster is indicated by a double asterisk, Asp25 by a triple asterisk and Pro333 by a white arrow. Tropomyosin (ribbon representations) shown in the B-state position lies over these residues, but is well-removed from them when it is in the M-state position, where interactions with residues Asp311 and Lys315 are likely (single asterisks in (c)). Note that the edges of the relatively flat interface over which tropomyosin moves is demarcated on one side by Asp25 on actin subdomain 1 and on the other by a cluster of amino acids (Thr229 to Leu236) on subdomain 4 (magenta diamond). Figures rendered using the program Chimera (Pettersen et al. 2004) based on data in Li et al. (2011) and Behrmann et al. (2012); (a,b) are modifications of Figure 2f and 2j in Li et al. (2011) with permission. The y-axis markers indicate the filament (and actin subunit) pointed end direction and the x-axis indicates the azimuthal direction traversed by tropomyosin from the M-state to the B-state across the relatively flat front facing interface of actin.

Figure 3

Maps of the interaction energy landscape between tropomyosin and F-actin. (a) Electrostatic Coulombic energy values were computed for a single tropomyosin placed super-helically on F-actin (Oda et al. 2009) at different axial and azimuthal positions (Orzechowski et al. 2012), as done previously for a similar but narrower search around the A-state position (Li et al. 2011). (a) The Coulombic landscape has a broad basin (blue) surrounding the energy minimum (indicated by +) (dark blue area) located close to the B-state position found by EM (0,0 position on the map). In contrast, the Coulombic energy maximum (indicated by ●) (yellow area) is close to the open M-state position of tropomyosin (Behrmann et al. 2012). Note: there is only one obvious energy minimum, and not three separate and discrete minima, which might represent B-, C- and M-state positions for tropomyosin on F-actin (cf. Lehman et al. 2000). (b) Number of oppositely charged pairs of residues on actin and tropomyosin making favorable electrostatic interactions at close range (< 5 Å) for each grid location in (a) were tabulated; the same conformers used in the map in (a) were examined in (b). Note the favorable basin in (a) corresponds to the region with greatest number of complementary charged residue pairs in (b). To compute these maps, actin-tropomyosin structures were first generated by sliding an atomic model of tropomyosin (Li et al. 2011) in longitudinal and azimuthal directions over actin in ±1 Å × 0.75° (~0.6 Å) increments to create a grid of tropomyosin locations. Intermediate positions were derived by interpolating/extrapolating between two actin-tropomyosin reference structures (+ and ●, mentioned above), then subjecting surface residues of F-actin and tropomyosin to an energy minimization. This removes steric clashes without significant changes in position of tropomyosin on F-actin. This procedure differs from that in Li et al. (2011), where only tropomyosin was minimized and F-actin was fixed, resulting in a small (~4°) azimuthal difference in the global minimum between the two determinations. All calculations used the CHARMM27 force-field (MacKerell et al. 1998, 2004; Brooks et al. 2009). Solvation was implemented according to the Generalized Born model and the GBSW protocol (Im et al. 2003) at 150 mM salt concentration.

Figure 4

Tropomyosin movement from B- to M-state. (a) The pseudo-atomic model of the actin-tropomyosin-myosin complex, obtained by fitting crystal structures into the cryo-EM density map of Behrmann et al. (2012), reveals that tropomyosin in the M-state fits snugly into a groove formed by actin and myosin. (b) Tropomyosin in the B-state (Li et al. 2011) (c) rotates slightly and shifts by ~23 Å to reach the M-state position (d), where tropomyosin is wedged (locked) between the myosin head and a bulge on actin subdomain 4 (Behrmann et al. 2012). Actin subunits are displayed in lavender, myosin heads in pale beige, and tropomyosin in the B- and M-states in yellow and blue respectively; tropomyosin residue R181 locations are highlighted in red.