Effect of Metal Cations on the Conformation of Myosin Subfragment-1-ADP-Phosphate Analog Complexes: A Near-UV Circular Dichroism Study
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- 作者：Y. Michael Peyser ; Katalin Ajtai ; Moshe M. Werber ; Thomas P. Burghardt ; and Andras Muhlrad
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：April 29, 1997
- 年：1997
- 卷：36
- 期：17
- 页码：5170 - 5178
- 全文大小：203K
- 年卷期：v.36,no.17(April 29, 1997)
- ISSN：1520-4995
文摘
The interaction of myosin with actin, coupled withhydrolysis of ATP, is the molecular basisof muscle contraction. The head segment of myosin, called S1,contains the distinct binding sites forATP and actin and is responsible for the ATPase activity. Themyosin-catalyzed ATP hydrolysis consistsof several intermediate steps and each step is accompanied byconformational changes in the S1 segment.The rate-limiting step of the ATP hydrolysis is the dissociationof the S1·ADP·Pi complex which isaccelerated by actin. The substitution of Pi withphosphate analogs (PA), such as vanadate, berylliumfluoride (BeFx) or aluminum fluoride(AlF4-), yields stable complexes which mimicthe intermediates ofthe ATP hydrolysis. In this work, tertiary structure changes in S1in the vicinity of aromatic residueswas studied by comparing near-UV circular dichroism (CD) spectra fromS1-nucleotide-phosphate analogcomplexes in the presence of Mg2+ and other cations.A significant difference between the MgATP andMgADP spectra indicated notable tertiary structural changesaccompanying the M**ADP·Pi 
M*ADPtransition. The spectra of theS1·MgADP·BeFx andS1·MgADP·AlF4- complexesresemble to thoseobtained upon addition of MgATP
S and MgATP to S1, and correspond tothe M*·ATP and M**·ADP·Piintermediates, respectively. We have found recently that thepresence of divalent metal cations (Me2+)is essential for the formation of stable S1·MeADP·PAcomplexes. Moreover, the nature of the metalcations strongly influences the stability of these complexes [Peyser,Y. M., et al. (1996) Biochemistry 35,4409-4416]. In the present work we studied the effect ofMg2+, Mn2+, Ca2+,Ni2+, Co2+, and Fe2+onthe near-UV CD spectrum of the ATP, ADP,ADP·BeFx, andADP·AlF4- containing S1 complexes.TheCD spectra obtained with ADP, ATPADP·BeFx andADP·AlF4- were essentially identicalin the presenceof Co2+ and rather similar in the case ofCa2+, while they were partially different in other cases.Aninteresting correlation was found between actin activation and ATPversus ADP difference spectra in thepresence of various metal ions. The distribution of the fractionalconcentration of the intermediates ofATP hydrolysis was estimated in the presence of each cation from the CDspectra with phosphate analogs.In the presence of Mg2+ the predominant intermediateis the M**·ADP·Pi state, which is inaccordancewith the kinetic studies. On the other hand with non-nativecations the predominant intermediate is theM*·ADP state and the release of ADP is the rate limiting step inthe myosin-catalyzed ATP hydrolysis.According to the results, the near-UV CD spectrum originating fromaromatic residues in S1 not only candistinguish identifiable states in the ATP hydrolysis cycle but canalso pinpoint to changes in the tertiarystructure caused by complex formation with nucleotide or nucleotideanalog and various divalent metalcations. These findings, that are correlative with actinactivation, and thus with the power stroke, suggestnew strategies for perturbing S1 structure in the continuous effortsdirected toward the elucidation of themechanism of muscle contraction.
M*ADPtransition. The spectra of theS1·MgADP·BeFx andS1·MgADP·AlF4- complexesresemble to thoseobtained upon addition of MgATPS and MgATP to S1, and correspond tothe M*·ATP and M**·ADP·Piintermediates, respectively. We have found recently that thepresence of divalent metal cations (Me2+)is essential for the formation of stable S1·MeADP·PAcomplexes. Moreover, the nature of the metalcations strongly influences the stability of these complexes [Peyser,Y. M., et al. (1996) Biochemistry 35,4409-4416]. In the present work we studied the effect ofMg2+, Mn2+, Ca2+,Ni2+, Co2+, and Fe2+onthe near-UV CD spectrum of the ATP, ADP,ADP·BeFx, andADP·AlF4- containing S1 complexes.TheCD spectra obtained with ADP, ATPADP·BeFx andADP·AlF4- were essentially identicalin the presenceof Co2+ and rather similar in the case ofCa2+, while they were partially different in other cases.Aninteresting correlation was found between actin activation and ATPversus ADP difference spectra in thepresence of various metal ions. The distribution of the fractionalconcentration of the intermediates ofATP hydrolysis was estimated in the presence of each cation from the CDspectra with phosphate analogs.In the presence of Mg2+ the predominant intermediateis the M**·ADP·Pi state, which is inaccordancewith the kinetic studies. On the other hand with non-nativecations the predominant intermediate is theM*·ADP state and the release of ADP is the rate limiting step inthe myosin-catalyzed ATP hydrolysis.According to the results, the near-UV CD spectrum originating fromaromatic residues in S1 not only candistinguish identifiable states in the ATP hydrolysis cycle but canalso pinpoint to changes in the tertiarystructure caused by complex formation with nucleotide or nucleotideanalog and various divalent metalcations. These findings, that are correlative with actinactivation, and thus with the power stroke, suggestnew strategies for perturbing S1 structure in the continuous effortsdirected toward the elucidation of themechanism of muscle contraction.