Does the S2 rod of myosin II uncoil upon two-headed binding to actin? A leucine-zippered HMM study.
Myosin II, like many molecular motors, is a two-headed dimer held together by a coiled-coil rod. The stability of the (S2) rod has implications for head-head interactions, force generation, and possibly regulation. Whether S2 uncoils has been controversial. To test the stability of S2, we constructed a series of "zippered" dimeric smooth muscle myosin II compounds, containing a high-melting temperature 32-amino acid GCN4 leucine zipper in the S2 rod beginning 0, 1, 2, or 15 heptads from the head-rod junction. We then assessed the ability of these and wild-type myosin to bind strongly via two heads to an actin filament by measuring the fluorescence quenching of pyrene-labeled actin induced by myosin binding. Such two-headed binding is expected to exert a large strain that tends to uncoil S2, and hence provide a robust test of S2 stability. We find that wild-type and zippered heavy meromyosin (HMM) are able to bind by both heads to actin under both nucleotide-free and saturating ADP conditions. In addition, we compared the actin affinity and rates for the 0- and 15-zippered HMMs in the phosphorylated "on" state and found them to be very similar. These results strongly suggest that S2 uncoiling is not necessary for two-headed binding of myosin to actin, presumably due to a compliant point in the myosin head(s). We conclude that S2 likely remains intact during the catalytic cycle.
Chakrabarty
Tania
T
Center for Biophysics and Computational Biology and Physics Department, University of Illinois, Urbana, Illinois 61801, USA.
Yengo
Chris
C
Baldacchino
Corry
C
Chen
Li-Qiong
LQ
Sweeney
H Lee
HL
Selvin
Paul R
PR
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D019281:Dimerization; D005822:Genetic Vectors; D016350:Leucine Zippers / Q000235:genetics; D008954:Models, Biological; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D018411:Spodoptera
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D024462:Myosin Type II