Extensively Methylated Myosin Subfragment-1: Examination of Local Structure, Interactions with Nucleotides and Actin, and Ligand-Induced Conformational Changes

Abstract

The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

“…The Mg 2+-and Ca 2+-ATPase activities of $1 were enhanced by methylation sixfold and three-fold, respectively, while the K+-EDTAATPase activity was almost abolished (10-15% of the control). These changes in ATPase activities are similar to those described in the literature (Bivin et al, 1994;Phan et al, 1994).…”

“…These our results are well consistent with recently published data of Phan and colleagues (1994) demonstrating that the methylafion, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on $1 by vanadate or beryllium fluoride, strongly impairs the coupling between the actin-and nucelotide-binding sites, and perturbs the spread of nucelotide-induced conformational changes throughout the $1 structure. The authors suggested that these methylation-caused changes in the $1 molecule are due to the methylation of Lys-83 (Phan et al, 1994), and our results support this assumption. On the other hand, the methylation-induced decrease of the $1 thermal stability is related, most likely, to the modification of lysine residues different from Lys-83.…”

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering

“…The pattern of inhibition of K+(EDTA)-ATPase and activation of divalent cation-dependent ATPase agrees with that previously reported by Kitagawa et al (1961) for reaction of a single lysyl E-NH2 group with 2,4,6-trinitrobenzenesulfonate and is typical of the effect of a number of myosin modifiers. The effect of methylation on ATPase activities reported here is also quite similar to several of those recently reported by Phan et al (1994); however, because the latter authors did not carry out an amino acid analysis of the methylated protein, it is difficult to make a direct comparison. When S1 [isolated from the central portion of the Q Sepharose peak (Fig.…”

“…It is known that dimethyllysine residues in proteins are not cleaved by trypsin at a significant rate, whereas reductive methylation of trypsin itself does not reduce its catalytic activity (Means, 1977). Although Phan et al (1994) reported increased susceptibility to heat denaturation after methylation of S1, reductive alkylation has been reported to stabilize chymotrypsinogen A (Fujita and Noda, 1991) and glycogen phosphorylase b (Shatsky et al, 1973) to denaturation, possibly by increasing the hydrophobicity of lysine E-amino groups. Resistance to proteolysis may have been a contributing factor in the successful crystallization of reductively methylated S1.…”

X-ray Structures of the Myosin Motor Domain of Dictyostelium discoideum Complexed with MgADP.cntdot.BeFx and MgADP.cntdot.AlF4-

“…It remained to be determined, however, whether the reductive alkylation had significantly altered the relative arrangement of domains within myosin. Recently, the use of the X-ray structure as a model for the contractile cycle has been questioned due to the changes in the enzymatic properties of the molecule induced by reductive methylation (Bivin et al, 1994;Phan et al, 1994).…”