Figure 1. Myosin-2 phylogeny, overall topology, and active site characteristics of human NM2C.

(A) Phylogenetic analysis divides human myosins-2 in the three subfamilies (i) nonmuscle and smooth muscle myosins-2, (ii) cardiac, (iii) and skeletal muscle myosins-2 (Foth et al., 2006). Nonmuscle myosin-2s are essential for the structural integrity of the cytoplasmic architecture during cell shape remodeling and motile events of eukaryotic cells, whereas all other myosins-2 play eminent roles in the contraction of smooth, cardiac and striated muscle cells (Sellers, 2000). Abbreviations used: NM2A: nonmuscle myosin-2A, NM2B: nonmuscle myosin-2B; NM2C: nonmuscle myosin-2C; SM: smooth muscle myosin-2; CardA: α-cardiac myosin-2; CardB: β-cardiac myosin-2; EO2: extraocular myosin-2; EMB: embryonic myosin-2; PERI: perinatal myosin-2; IIb: fast skeletal muscle myosin-2; IIx/d: skeletal muscle myosin-2; IIa: slow skeletal muscle myosin-2. (B) Architecture of the crystallized NM2C construct in the pre-powerstroke state. The myosin motor domain and the α-actinin repeats are shown in cartoon representation in green and grey color. The nucleotide is shown in spheres representation. Inset, Conserved key residues that interact with the nucleotide in the NM2C active site. The F_o-F_c omit map of Mg²⁺·ADP·VO₄ is contoured at 4σ. The salt bridge between switch-1 R261 and switch-2 E483 is highlighted. (C) Subdomain architecture of NM2C. The U50 kDa is shown in green, the L50 kDa in purple, the converter in grey, and the Nter in blue. The region shown in orange corresponds to the active site and the junction of U50 kDa and L50 kDa. The bound nucleotide is shown in spheres representation. The location of the SH1-SH2 helix and the relay helix in the L50 kDa is highlighted.

Figure 1—figure supplement 1. Myosin-2 ATPase cycle, expression constructs, and structural alignment of the NM2C C_α coordinates.

(A) Consensus scheme of the myosin and actomyosin ATPase cycle. The upper part represents the myosin (M) ATPase cycle. The lower part represents the ATPase cycle in the presence of F-actin (A). The asterisk denotes enhanced states of the intrinsic myosin fluorescence, which are attributed to the nucleotide-induced changes in the microenvironment of the conserved relay loop W525. Lowercase k denotes a rate constant. k_+D=k₊₆, k_-D = k_-6, k_+AD = k₊₆, k_-AD = k_-6. An uppercase K denotes a dissociation equilibrium constant (K = k_-x/k_+x) throughout this work. Normal and bold face notation denote the respective kinetic constants in the absence and presence of F-actin. The main pathway of the actomyosin ATPase cycle is highlighted in grey. Strong and weak actin binding states are indicated. (B) Schematic representation of the expression constructs used in this study. Top, Nonmuscle myosin-2C contains a N-terminal motor domain (green) followed by a neck and a tail domain (dark grey). Middle, For kinetic studies of NM2C, R788K, and R788E, the motor domain was directly fused to spectrin repeats 1 and 2 from Dictyosteliumα-actinin (light grey) which serves as an artificial lever arm. The concept of the artificial lever arm has been successfully used in structural and kinetic studies of myosins-2 (Heissler and Manstein, 2011; Furch et al., 1999; Kliche et al., 2001; Münnich et al., 2014). Bottom: For structural studies, the N-terminal 45 amino acids were deleted. The numbering refers to the amino acid sequence of the full-length protein. (C) The NM2C C_α atoms (green) in ribbon representation superimpose with a root mean square deviation (r.m.s.d.) of 0.57 Å to chicken smooth muscle myosin-2 (grey, PDB entry 1BR2), with 0.78 Å to scallop striated muscle myosin-2 (orange, PDB entry 1QVI), and 0.73 Å to Dictyostelium nonmuscle myosin-2 (blue, PDB entry 2XEL), underlining a strong correlation between C_α geometry and overall motor domain fold. The JK-loop and the nucleotide are highlighted in color and spheres representation. (D) Relative orientation of converter and lever in NM2C (green), chicken smooth muscle myosin-2 (PDB entry 1BR2, grey), and scallop striated muscle myosin-2 (PDB entry 1QVI (orange), 1DFL (violet)) motor domain structures in cartoon representation. α-helices are depicted as cylinders.