Structural basis for actomyosin chemomechanical transduction by non-nucleoside triphosphate analogues.
Methylation of 2-[(2,4-dinitrophenyl)amino]ethyl triphosphate (dNOTP) was found to abolish its ability to support actin sliding in the in vitro motility assay. A comparative study of the interaction of myosin subfragment 1 (S1) and actoS1 with methylated (MdNOTP) and non-methylated dNOTP was undertaken. Both analogues were shown to be substrates for S1 NTPase in the presence of K+/EDTA, Ca2+, or Mg2+, although their rates of hydrolysis in the presence of the divalent cations were significantly greater than that occurring for ATP. However, actin had only a marginal effect on the rate of hydrolysis of MdNOTP, in sharp contrast to its effect on the hydrolysis of dNOTP and ATP which were quite similar. Moreover, while dNODP is able to form stable ternary S1 complexes with orthovanadate (Vi) or berylium fluoride (BeFx), whose formation results in increased thermal stability of S1, the methylated diphosphate analogue was unable to do so. These differences can be related to methylation-induced changes in the conformation of dNOTP indicated by molecular-modeling approaches. These studies suggest that methylation prevents the specific interaction of the aryl ring of dNOTP with S1 in the adenine binding region necessary for the formation of the force-producing intermediate (M. D. P*) during the S1 Mg(2+)-NTPase cycle.
Gopal
D
D
Department of Biology, College of Arts and Sciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Bobkov
A A
AA
Schwonek
J P
JP
Sanders
C R
CR
Ikebe
M
M
Levitsky
D I
DI
Burke
M
M
D017766:Acid Anhydride Hydrolases; D000199:Actins / Q000494:pharmacology; D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000032:analysis; D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002152:Calorimetry, Differential Scanning; D006868:Hydrolysis / Q000187:drug effects; D008956:Models, Chemical; D009068:Movement; D015879:Myosin Subfragments / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
C095929:2-((2,4-dinitrophenyl)amino)ethyl triphosphate; C095930:2-(methyl(2,4-dinitrophenyl)amino)ethyl triphosphate; D000199:Actins; D000814:Aniline Compounds; D015879:Myosin Subfragments; D009943:Organophosphorus Compounds; D000205:Actomyosin; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase