Addition of lysines to the 50/20 kDa junction of myosin strengthens weak binding to actin without affecting the maximum ATPase activity.
Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K(m) for actin-activated ATPase activity decreased 15-fold, but V(max) was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V(max). The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin- and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.
Joel
Peteranne B
PB
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
Sweeney
H Lee
HL
Trybus
Kathleen M
KM
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004789:Enzyme Activation; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship
D000199:Actins; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008239:Lysine