Crystal structures of human cardiac beta-myosin II S2-Delta provide insight into the functional role of the S2 subfragment

Abstract

Myosin II is the major component of the muscle thick filament. It consists of two N-terminal S1 subfragments ("heads") connected to a long dimeric coiled-coil rod. The rod is in itself twofold symmetric, but in the filament, the two heads point away from the filament surface and are therefore not equivalent. This breaking of symmetry requires the initial section of the rod, subfragment 2 (S2), to be relatively flexible. S2 is an important functional element, involved in various mechanisms by which the activity of smooth and striated muscle is regulated. We have determined crystal structures of the 126 N-terminal residues of S2 from human cardiac beta-myosin II (S2-Delta), of both WT and the disease-associated E924K mutant. S2-Delta is a straight parallel dimeric coiled coil, but the N terminus of one chain is disordered in WT-S2-Delta due to crystal contacts, indicative of unstable local structure. Bulky noncanonical side chains pack into a/d positions of S2-Delta's N terminus, leading to defined local asymmetry and axial stagger, which could induce nonequivalence of the S1 subfragments. Additionally, S2 possesses a conserved charge distribution with three prominent rings of negative potential within S2-Delta, the first of which may provide a binding interface for the "blocked head" of smooth muscle myosin in the OFF state. The observation that many disease-associated mutations affect the second negatively charged ring further suggests that charge interactions play an important role in regulation of cardiac muscle activity through myosin-binding protein C.

Fig. 1.

Localization of S2-Δ in myosin II and comparison of various S2-Δ sequences. (A) Organization of the myosin II molecule. S1, subfragment 1 containing the ATPase domain (dark blue) and the regulatory domain consisting of lever arm (light blue) and essential (ELC) and regulatory light chain (RLC, shown in dark and light green, respectively); S2, subfragment 2 with S2-Δ highlighted in red; LMM in gray. The sketch is to approximate scale only. (B) Alignment of the N termini of several representative S2 sequences. The sequence numbering refers to S2-Δ. Acidic and basic residues are shaded in red and blue, respectively. Noncanonical residues in a/d positions of the coiled coil are shown in italicized bold characters. a/d positions occupied by large noncanonical residues in the majority of sequences shown are indicated by green shades of the coiled-coil ruler (cc). Black boxes show the three negative charge rings of S2-Δ (E894–D906, E921–E935, and E944–E958). Positions of previously identified FHC amino acid substitutions (FHC) are indicated by a star (∗). The sequences represent myosin heavy chains from vertebrates and invertebrates, different tissue and with different regulatory properties. S2-Δ, human cardiac β-myosin II (MYH7_HUMAN; UniProtKB accession no. P12883); gg_cardiac, chicken cardiac (MYSC_CHICK; P29616); hs_skeletal, human skeletal (MYH13_HUMAN, Q9UKX3); gg_skeletal, adult chicken skeletal (MYSS_CHICK, P13538); hs_smooth, human smooth muscle (MYH11_HUMAN, P35749); gg_smooth, chicken gizzard (MYH11_CHICK, P10587); ce_unc54, Caenorhabditis elegans body wall muscle (MYO4_CAEEL; P02566); aa_striated, Aequipecten irradians (scallop) striated muscle (MYS_AEQUIR, P24733). This figure was prepared with ESPript (33).

Fig. 2.

Ribbon plots of the asymmetric unit of (A) WT-S2-Δ and (B) E924K-S2-Δ. N designates the N terminus. Light-gray ribbons depict crystallographic neighbors. In A, dark-gray spheres show the position of two mercury atoms bound to C905 and C947. The pink helical coil indicates the position of residual electron density at the flexible N terminus of the chain in magenta. All molecular representations were prepared with PyMOL (34).

Fig. 3.

Local asymmetry in S2-Δ. (A) Hydrogen-bonding network at the N terminus of E924K-S2-Δ. (B) Asymmetric hydrogen bonding network around R869 shown from “front” and “back” of WT-S2-Δ. (C) Asymmetry of N-terminal noncanonical a/d residues of E924K-S2-Δ. The dashed lined connects the center of the two α-helices and thus defines the expected twofold dimer axis. (D) Local stagger along WT- and E924K-S2-Δ, calculated from trace positions determined with TWISTER (35). (E) Kink at M877 in WT-S2-Δ.

Fig. 4.

Model of the OFF state, based on the structure of S2-Δ, a model of smooth muscle myosin in the 10S conformation (29) and the cryo-EM reconstruction of tarantula thick filament (2). The model was constructed by introducing a single bent at position 877 into S2-Δ. The electrostatic surface potential of S2-Δ was calculated with APBS (36). The blocked head is shown in yellow with the amino acids framing loop 2 shown in magenta, although the actual residues of loop 2 are missing. The remaining residues of the approximate actin-binding interface have been colored in dark green (37). (Inset) Magnification of the interaction after a clockwise 90° rotation around the coiled-coil main axis. For an unobstructed view, the free head has been omitted.