Table 2. Structure function relationships in the myosin-2 motor domain.

Interactions between residues in the active site involved in nucleotide binding and release kinetics based on this work and previous biochemical and structural studies on myosin motor domains (Miller et al., 2007; Risal et al., 2004; Swank et al., 2006; Grammer et al., 1993; Szilagyi et al., 1979). It is of note that myosin is a highly allosteric enzyme and nucleotide binding and release kinetic involve numerous interactions and subtle structural rearrangements of residues from different motor subdomains. Kinetic parameters from monomeric myosin motor domain constructs that are associated with a structural interaction are listed for direct comparison. An emerging trend from this analysis is that the myosin-2 kinetic cycle does not have a selectivity of ATP versus ADP. The presence of F-actin results in different allosteric communication pathways in myosins-2 and establishes ATP/ADP binding selectivity. Overall, nucleotide-binding rates are decreased for the group of nonmuscle and smooth muscle myosins-2 compared to myosins-2 from cardiac and skeletal muscle. The lacking salt bridge interactions between JK-loop, U50 kDa and switch-1 in nonmuscle myosins-2 results in decreased second-order binding rate constants for ATP (K₁k₊₂) and ADP (k_+D) (Figure 1—figure supplement 1A). Either a salt bridge interaction or hydrophobic interactions between the A-loop and the P-loop of muscle and cardiac myosins-2 at the active site favor fast nucleotide binding kinetics and does not or only marginally discriminate between ADP and ATP. The lack of a salt bridge interactions can have different effects dependent on the coordinating residue in the A-loop: An asparagine in the A-loop of nonmuscle myosins-2A and −2B favors ADP over ATP binding to actomyosin. A glutamine in the NM2C A-loop, which has a longer side chain than asparagine, abolishes ATP/ADP sensitivity in NM2C and the closely related smooth muscle myosin-2. The number of salt bridge interactions between P-loop, switch-1, and the Nter correlates with the thermodynamic and kinetic coupling, and the actin-activated ADP release rates in all myosins-2. Abbreviations used: NM2A: human nonmuscle myosin-2A; human NM2B: nonmuscle myosin-2B (PDB entry 4PD3); NM2C: human nonmuscle myosin-2C; SM: chicken smooth muscle myosin-2 (PDB entry 1BR2); CARD: human β-cardiac myosin-2 (PDB entry 4DB1); Oc ST: rabbit striated muscle myosin-2 (PDB entry 1DFL).

Myosin	Residue	Residue	Residue	Residue	Parameter
	JK-loop	U50 kDa	JK-loop	Switch-1	K1k+2	K+D
Hs NM2A (Kovács et al., 2003)	D315	R272	G312	D230	0.56	0.55
Hs NM2B (Wang et al., 2003)	D322	R279	G319	D237	0.65	0.81
Hs NM2C	E341	C299	G339	D257	0.48	0.39
Gg SM (Cremo and Geeves, 1998)	D328	R285	A325	D243	2.1	1.8
Hs CARD (Deacon et al., 2012)	D325	R281	S322	D239	1.5	1.5
Oc ST (Ritchie et al., 1993; Kurzawa-Goertz et al., 1998)	D323	R280	N321	N238	3.9	1.7
	Residue	Residue	Parameter
	A-loop	P-loop	k+AD	K1k+2	k+D	K1k+2	k+AD/ K1k+2
Hs NM2A (Kovács et al., 2003)	N126	E182	2.72	0.14	0.55	0.56	19.4
Hs NM2B (Wang et al., 2003)	N130	E186	2.41	0.24	0.81	0.65	10
Hs NM2C	Q150	E206	2.54	1.86	0.39	0.48	1.5
Gg SM (Cremo and Geeves, 1998)	Q129	E185	3.6	2	1.8	2.1	4.4
Hs CARD (Deacon et al., 2012)	W130	V186	4.4	1.1	1.5	1.5	1.6
Oc ST (Ritchie et al., 1993; Kurzawa-Goertz et al., 1998)	R128	E184	1.6	2.5	1.7	3.9	1.6
	Residue	Residue	Residue	Parameter
	P-loop	Switch-1	Nter	KAD/KD	k-AD/k-D	k-AD
Hs NM2A (Kovács et al., 2003)	E175	K228	H676	0.7	2.8	1.72
Hs NM2B (Wang et al., 2003)	E179	K235	H683	0.2	0.7	0.35
Hs NM2C	E199	K255	H700	0.11	1	0.65
Gg SM (Cremo and Geeves, 1998)	E178	K241	H689	4.2	12	15
Hs CARD (Deacon et al., 2012)	E179	R237	E677	42	103.3	150
Oc ST (Ritchie et al., 1993; Kurzawa-Goertz et al., 1998)	E177	R236	E675	49	250	500