MgADP-induced changes in the structure of myosin S1 near the ATPase-related thiol SH1 probed by cross-linking.
The structural consequences of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca2(+)-ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-pre-modified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [14C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa "domain" which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM.
Rajasekharan
K N
KN
Department of Biology, Case Institute of Technology, Case Western Reserve University, Cleveland, Ohio 44106.
Mayadevi
M
M
Agarwal
R
R
Burke
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D015879:Myosin Subfragments / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D008301:Maleimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D009218:Myosins; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D005033:Ethylmaleimide; C023860:N,N'-2-phenylenedimaleimide