X-ray Structures of the Apo and MgATP-bound States of Dictyostelium discoideum Myosin Motor Domain (original) (raw)
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Biochemistry, 1995
The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin I1 complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-M ADP complex belong to space group P21212 with unit cell parameters of a = 105.3 A, b = 182.6 1 , and c = 54.7 A, whereas the crystals of the aluminum fluoride com lex belong to the orthorhombic space group C2221 with unit cell obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADPBeF, is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADPeAlF4-exhibits significant domain movements. The structures suggest that the MgADPBeF, complex mimics the ATP bound state and the MgADPoAlF4-complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADPAlF4-complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain. dimensions of a = 87.9 A, b = 149.0 !i , and c = 153.8 A. I Abbreviations: myosin S 1, myosin subfragment-I; S lDc, Dicryostelium discoideum myosin I1 motor domain; PMSF, phenylmethanesulfonic acid; S 1 DcBeF,.MgADP, the beryllium fluoride-ADP complex of D . discoideum myosin head; SlDcAIF4*MgADP, the aluminum fluoride-ADP complex of D. discoideum myosin head; BTP, Bis-Trispropane; DT", dithiothreitol; HEPES, N-(2-hydroxyethyl)piperazine-K-2-ethanesulfonic acid; rms, root mean square; Pi, phosphate; EDTA, ethylenediaminetetraacetic acid. 0006-2960/95/0434-8960$09.00/0 , -. ,
The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin I1 complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-M ADP complex belong to space group P21212 with unit cell parameters of a = 105.3 A, b = 182.6 1 , and c = 54.7 A, whereas the crystals of the aluminum fluoride com lex belong to the orthorhombic space group C2221 with unit cell obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADPBeF, is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADPeAlF4-exhibits significant domain movements. The structures suggest that the MgADPBeF, complex mimics the ATP bound state and the MgADPoAlF4-complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADPAlF4-complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain. dimensions of a = 87.9 A, b = 149.0 !i , and c = 153.8 A. I Abbreviations: myosin S 1, myosin subfragment-I; S lDc, Dicryostelium discoideum myosin I1 motor domain; PMSF, phenylmethanesulfonic acid; S 1 DcBeF,.MgADP, the beryllium fluoride-ADP complex of D . discoideum myosin head; SlDcAIF4*MgADP, the aluminum fluoride-ADP complex of D. discoideum myosin head; BTP, Bis-Trispropane; DT", dithiothreitol; HEPES, N-(2-hydroxyethyl)piperazine-K-2-ethanesulfonic acid; rms, root mean square; Pi, phosphate; EDTA, ethylenediaminetetraacetic acid. 0006-2960/95/0434-8960$09.00/0 , -. ,
Journal of muscle research and cell motility, 1997
Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP....
Kinetic characterization of a cytoplasmic myosin motor domain expressed in Dictyostelium discoideum
Proceedings of the National Academy of Sciences, 1993
A detailed kinetic study of the interaction of a recombinant myosin head fagment (MHF) of Dictyostelium discoideun with actin and adenine nucleotides has been made by using a combination of rapid-reaction, equilibrium, and fluorescence methods. MHF is equivalent in size to a proteolytic faent of skeletal muscle myosin, subfragment 1 (Si), the simplest unit of myosin to retain enzymatic and functional activity. The results show that qualitatively the interactions of MHF with nucleotides and actin are the same as those of S1.
Dictyostelium discoideum Myosin II: Characterization of Functional Myosin Motor Fragments †
Biochemistry, 1997
The transient kinetic properties of the recombinant myosin head fragments M761 and M781, which both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781, and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding, and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, [16056][16057][16058][16059][16060][16061][16062][16063][16064]. We further show that C-terminal extension of M761 with one or two R-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and function of the myosin motor. † Abstract published in AdVance ACS Abstracts, December 15, 1996. 1 Abbreviations: ELC, essential light chain; LCBD, light chain binding domain; mantADP, 2′(3′)-O-(N-methylanthraniloyl)adenosine 5′-diphosphate; MHF, myosin head fragment; pyr-actin, pyrene-labeled actin; RLC, regulatory light chain; S1, myosin subfragment 1; ul-actin, unlabeled actin.
Crystal structure of the motor domain of a class-I myosin.
The crystal structure of the motor domain of Dictyostelium discoideum myosin-IE, a monomeric unconventional myosin, was determined. The crystallographic asymmetric unit contains four independently resolved molecules, highlighting regions that undergo large conformational changes. Differences are particularly pronounced in the actin binding region and the converter domain. The changes in position of the converter domain reflect movements both parallel to and perpendicular to the actin axis. The orientation of the converter domain is approximately 30 degrees further up than in other myosin structures, indicating that MyoE can produce a larger power stroke by rotating its lever arm through a larger angle. The role of extended loops near the actin-binding site is discussed in the context of cellular localization. The core regions of the motor domain are similar, and the structure reveals how that core is stabilized in the absence of an N-terminal SH3-like domain.
Dictyostelium discoideum Myosin II: Characterization of Functional Myosin Motor Fragments
Biochemistry, 1997
The transient kinetic properties of the recombinant myosin head fragments M761 and M781, which both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781, and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding, and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two R-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and function of the myosin motor.