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.
Center for Biophysics and Computational Biology and Physics Department, University of Illinois, Urbana, Illinois 61801, USA.
Tania
T
Chakrabarty
Chris
C
Yengo
Corry
C
Baldacchino
Li-Qiong
LQ
Chen
H Lee
HL
Sweeney
Paul R
PR
Selvin
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