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. 2017 Dec 19;6:e32742. doi: 10.7554/eLife.32742

Figure 2. Conformational changes of the JK-loop in the myosin active site.

(A) Top view on the NM2C active site in the pre-powerstroke state (green) superimposed on pre-powerstroke state structures from chicken smooth muscle myosin-2 (grey, PDB entry 1BR4), Dictyostelium nonmuscle myosin-2 (blue, PDB entry 2XEL), and scallop striated muscle myosin-2 (orange, PDB entry 1QVI). The ATP analog ADP⋅VO4 is shown in spheres representation. (B) Conformation of the JK-loop in vicinity to the NM2C active site. The JK-loop flanks the active site and connects helices J and K. The distance between the residue Q340 of the JK-loop in the U50 kDa and the D257 of switch-1 of the active site is ~8.8 Å. The distance between residue S336 of the JK-loop and switch-1 D257 is ~7.4 Å. Switch-1 residue N256 interacts with α-phosphate (3.1 Å) and β-phosphate (3.5 Å) group of ADP⋅VO4 in the active site. NM2C is colored in green/orange, the JK-loop is colored in brick red and ADP⋅VO4 is shown in spheres. (C) Interactions between the JK-loop and the switch-1 region are compared between the NM2C (green) and scallop striated muscle myosin-2 (orange, PDB entry 1QVI). A-loop residue R128 is coordinating the interaction to the ADP adenosine in the active site of striated muscle myosin-2. The distance between the residues is 3.2 Å. R128 further forms a hydrogen bond (2.8 Å) with E184 of the P-loop. JK-loop N321 is in hydrogen bond interaction with switch-1 N238, located at a distance of 4.6 Å to the hydroxyl group of the C2’ of the ADP ribose. The connectivity between switch-1 and the nucleotide is further strengthened by a hydrogen bond between N237 and the ADP ribose. NM2C lacks all interactions described for scallop striated muscle myosin-2 due to the replacement of R128 with Q150 and JK-loop shortening which increases the distance to the adenosine in the active site to 5.8 Å and disrupts constrains between swich-1 and the JK-loop. All residues in the JK-loop region are labeled for scallop striated muscle myosin-2 (PDB entry 1QVI). For NM2C only amino acid substitutions are labeled for legibility. (D) Superimposition of the NM2C pre-powerstroke state structure (green) and the actin-bound near-rigor actoNM2C complex (red) shows that the nucleotide-binding site does not undergo major structural changes. Actin subunits are colored in shades of grey and the nucleotide is shown in spheres representation. (E) Sequence alignment of select structural elements in the myosin motor domain that interact with the JK-loop. Interactions of A-loop R128 are highlighted with brackets for scallop striated muscle myosin-2 (PDB entry 1QVI). All highlighted interactions are absent in NM2C due to the presence of Q150 in the A-loop. Abbreviations used: Hs NM2C: human NM2C (NP_079005.3); Hs NM2A: human nonmuscle myosin-2A (NP_002464.1); NM2B: human nonmuscle myosin-2B (NP_005955.3); Gg SM: chicken smooth muscle myosin-2 (NP_990605.2); Hs CARD: human beta β-cardiac muscle myosin-2 (NP_000248.2); Ai ST: scallop striated muscle myosin-2 (P24733.1). PDB entries are indicated when available.

Figure 2.

Figure 2—figure supplement 1. Active site characteristics in myosin-2 motor domains.

Figure 2—figure supplement 1.

(A) Sequence alignment of myosin-2 JK-loops. The asterisk indicates the invariant, negatively charged residues corresponding to NM2C E341. The abbreviations used are as follows: Hs NM2C: human NM2C (NP_079005.3); Hs NM2A: human nonmuscle myosin-2A (NP_002464.1); NM2B: human nonmuscle myosin-2B (NP_005955.3); Gg SM: chicken smooth muscle myosin-2 (NP_990605.2); Hs CARD: human beta β-cardiac muscle myosin-2 (NP_000248.2); Ai ST: scallop striated muscle myosin-2 (P24733.1); Dd NM2: Dictyostelium nonmuscle myosin-2: (XP_637740.1); Dm EMB: Drosophila embryonic body wall muscle myosin-2 (P05661 with spliced exon 7a); Dm IFI: Drosophila indirect flight muscle myosin-2 (P05661 with spliced exon 7d). PDB entries are indicated when available. Cardiomyopathy-associated mutations in β-cardiac myosin-2 on positions F312 (F312C), V320 (V320M), A326 (A326P), and E328 (E328G) are highlighted in the boxed areas. (B) Close-up view of the active site region of pre-powerstroke state structures of NM2C (green), chicken smooth muscle myosin-2 (grey, PDB entry 1BR2), and Dictyostelium nonmuscle myosin-2 (blue, PDB entry 2XEL). The distance between the JK-loops of smooth muscle myosin-2 and Dictyostelium nonmuscle myosin-2 and switch-1 is ~2.8 Å and 8.8 Å for NM2C. The ADP⋅VO4 complex in the active site is shown in spheres. (C) Close-up view of the active site region of pre-powerstroke state chicken smooth muscle myosin-2 (grey, PDB entry 1BR2). The distance between residue P324 of the JK-loop (yellow) in the U50 kDa and D243 of switch-1 is ~2.8 Å. Switch-1 residue N242 interacts with α-phosphate (2.8 Å) and β-phosphate (3.1 Å) group of ADP⋅ALF4 (shown in spheres) in the active site. (D) The volume of the active site, indicated by the spheres, was determined to 3065 Å3 in NM2C by fitting a sphere with a radius (r) of 9.012 Å to the active site with UCSF Chimera (Pettersen et al., 2004). The volume of the sphere was calculated based on its radius using the equation volume = 4/3πr3. (E) The volume of the active site of scallop striated muscle myosin-2 (PDB entry 1QVI) was determined to 697 Å3 based on a radius of r = 5.5 Å as in (D). The structures shown in (D) and (E) are in the pre-powerstroke state.