Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex

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Abstract
Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1) to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied and Morales (1988) Proc. Nat. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido) fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5''-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[[4-(dimethaylamine)phenyl]azo]phenyl]maleimide or N-[[(iodacetyl)-amino]ethyl]-5-napthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the flourescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 .ANG.; Cys-697 (S1), 49.4 .ANG.; active site of S1, .gtoreq. 44 .ANG. ; nucleotide binding site (actin), 41.1 .ANG. and Cys, 374 (actin), .apprx. 53 .ANG.. Addition of MgATP had a small effect on the distance between the peptide and Cys-707, increasing it by .apprx. 2%, while it had a more pronounced effect on the distance between the peptide and Cys-697, where an increase of 14% was observed. The effect of actin on these two distances was negligible. These data enabled us (1) to place for the first time an interprotein contact in the three-dimensional map of the acto-S1 complex, (2) to definitively exclude models of communication between the nucleotide and actin binding site in S1 that involved direct contact or close proximity of these two loci of S1, and (3) to detect intraprotein motion in S1 induced by binding of MgATP to the protein.