Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment-1

Abstract

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.

Effect of Nucleotides and Actin on the Intramolecular Crosslinking of Myosin Subfragment-1

“…In conclusion, the 27-and 20-kDa fragments, which are far away from each other in the SI primary sequence, are at least partially proximal in the spatial structure of the molecule. This proximity has also been shown by the recently described atomic structure of SI (Rayment et al, 1993a 27-kDa fragment in addition to the regions described earlier by Wong (1989) andRajasekharan et al (1989), which makes the cross-linking of these regions possible.…”

“…The formation of the 27-kDa/20-kDa products in the reactions with the four cross-linkers used in our study was not affected by MgATP. Similarly, no significant nucleotide effect was seen on the cross-linking of the SHi thiol with benzophenone iodoacetamide to Glu-88 (Lu et al, 1986;Lu & Wong, 1989) or with 4-((V-maleimido)benzophenone to a region at 14-16 kDa from the N-terminus on the 27-kDa fragment (Rajasekharan et al, 1989). Actin inhibits the formation of the 27-kDa/20-kDa cross-link with EEDQ, but has no effect with the three other cross-linkers used in this study.…”

“…It was shown by these studies that the distance between SH) (Cys-707) and SH2 (Cys-697) [numbering is according to Tong and Elzinga (1983)] changes under the influence of nucleotide binding (Burke & Reisler, 1977;Dalbey et al, 1983). In the absence of nucleotide, the SHi thiol was cross-linked to Glu-88, which is near the N-terminus, while in the presence of nucleotide it also cross-linked to a region located 57-60 kDa away from the N-terminus of SI (Lu & Wong, 1989). On the other hand, the SH2 thiol cross-links by 4,-maleimidobenzophenone to Arg 239 at the N-terminal region of the central domain in the absence of MgADP and, in its presence, the binding shifts to the direction of the C-terminus (Rajasekharan etal., 1989;Agarwal et al, 1991).…”

Nonmuscle Myosin Heavy Chain IIA Mutations Define a Spectrum of Autosomal Dominant Macrothrombocytopenias: May-Hegglin Anomaly and Fechtner, Sebastian, Epstein, and Alport-Like Syndromes

“…Judging from amino acid substitutions on a well-characterized synthetic peptide that forms an ahelical polypeptide (O'Neil and DeGrado 1990), the mutations to histidine at both the R702 and R705 positions would result in an energy difference of 0.62 kcal/mole in the a-helix, thus destabilizing the secondary structure. Myosin cross-linking studies have biochemically confirmed these potential interactions and the close proximity of the residues within the domain depicted in figure 4a and c (Lu and Wong 1989). Lastly, modifications in the region of the two cysteines defining the functionality of this helical region have been shown to have severe biochemical ramifications, by altering nucleotide exchange, ATPase activity, and actin sliding activity (Tiepold et al 2000).…”

Myosin Catalytic Domain Flexibility in MgADP

“…Internal fluctuations of myosin heads induced by nucleotide binding have been inferred previously from changes of sulfhydryl reactivity (Burke & Reisler, 1977;Schaub et al, 1975), cross-linking studies (Wells & Yount, 1979; Lu & Wong, 1989), NMR (Shriver & Sykes, 1982), and EPR (Barnett & Thomas, 1987). Most of these studies were confined to isolated myosin in solution and were not suited to resolving or observing the heterogeneity of the structures within a complex of myosin and nucleotide.…”