Functional role of loop 2 in myosin V.
Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate-limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M.ADP.Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Christopher M
CM
Yengo
H Lee
HL
Sweeney
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002645:Chickens; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D024701:Myosin Type V / Q000737:chemistry* / Q000235:genetics; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011721:Pyrenes / Q000737:chemistry; D040681:Structural Homology, Protein
D000199:Actins; D010710:Phosphates; D011721:Pyrenes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; C030984:pyrene; D000251:Adenosine Triphosphatases; D024701:Myosin Type V