Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.
Recent crystallographic studies have suggested structural differences between the complexes of S1.Mg.ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO(4)3-) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1.ADP.BeFx, S1.ADP. AlF4- and S1.ADP-VO(4)3- complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20 degrees C lead to the conclusion that S1.ADP.BeFx is similar to S1*.ATP, while S1.ADP.AlF4- and S1.ADP.VO(4)3- are more similar to S1**.ADP.Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N'-p-phenylene dimaleimide) S1.ADP[C2H2].AlF4- and S1.ADP[C2H2]. BeFx. Our results suggest that the conformations of the S1.ADP.AlF4-, S1.ADP.VO(4)3- and S1.ADP.BeFx complexes in the Cys707-Cys697 region are distinct from each other, with the former two at least partially resembling the S1**.ADP.Pi state, while the latter is similar to the prehydrolyzed S1*.ATP state.
Phan
B C
BC
Department of Chemistry and Biochemistry, University of California, Los Angeles, USA.
Peyser
Y M
YM
Reisler
E
E
Muhlrad
A
A
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D003374:Coumarins / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry* / Q000187:drug effects; D005456:Fluorescent Dyes; D015879:Myosin Subfragments / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects*; D011817:Rabbits; D013439:Sulfhydryl Reagents
D003374:Coumarins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D010710:Phosphates; D013439:Sulfhydryl Reagents; D000244:Adenosine Diphosphate; C006421:1,5-I-AEDANS; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D003545:Cysteine