Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41

Kim, E.; Motoki, Masao; Seguro, Katsuya; Mühlrád, András; Reisler, Emil

doi:10.1016/s0006-3495(95)80072-x

Cited by 59 publications

(79 citation statements)

References 32 publications

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“…The smaller domain can be further divided into subdomains 1 and 2, and the larger domain consists of subdomains 3 and 4. Proteolysis studies and fluorescence resonance energy transfer assays reveal shifts in the position of subdomain 2 in ATP vs. ADP monomeric actin (9)(10)(11). Consistent with this finding, three-dimensional reconstructions of electron micrographs of actin filaments revealed a disordering of subdomain 2 in ADP compared with ADPBeF actin filaments (12,13).…”

supporting

confidence: 64%

A change in actin conformation associated with filament instability after P _i release

Belmont

Orlova

Drubin

1999

Proc. Natl. Acad. Sci. U.S.A.

155

147

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The ability of actin to both polymerize into filaments and to depolymerize permits the rapid rearrangements of actin structures that are essential for actin's function in most cellular processes. Filament polarity and dynamic properties are conferred by the hydrolysis of ATP on actin filaments. Release of inorganic phosphate (P i ) from filaments after ATP hydrolysis promotes depolymerization. We identify a yeast actin mutation, Val-159 to Asn, which uncouples P i release from the conformational change that results in filament destabilization. Three-dimensional reconstructions of electron micrographs reveal a conformational difference between ADP-P i filaments and ADP filaments and show that ADP V159N filaments resemble ADP-P i wild-type filaments. Crystal structures of mammalian ␤-actin in which the nucleotide binding cleft is in the ''open'' and ''closed'' states can be used to model actin filaments in the ADP and ADP-P i conformations, respectively. We propose that these two conformations of G-actin may be related to two functional states of F-actin.Regulation of actin filament dynamics is essential for cytokinesis, cell motility, and control of cell shape and polarity. The ATP cycle of actin is fundamental to this regulation. Actin polymerizes preferentially from ATP-bound actin, and this bound ATP is hydrolyzed after assembly. The subsequent release of inorganic phosphate (P i ) destabilizes the filament and promotes actin filament disassembly (1, 2). The addition of excess P i causes actin to behave as an equilibrium polymer, with the same critical concentration at both ends of the filament (3, 4). Beryllium fluoride and aluminum fluoride also can stabilize actin filaments by binding to the P i site and mimicking ADP-P i or ATP filaments (5, 6).The different assembly properties of ADP versus ATP or ADP-P i actin suggests that there is a conformational change in actin after P i release. Much has been learned about the nucleotide hydrolysis-dependent conformational changes that occur in G proteins and motor proteins (for review see ref. 7). However, the crystal structures of rabbit muscle actin in the ADP-and ATP-bound states show only minor structural differences (8). Actin has two major domains separated by a nucleotide binding cleft. The smaller domain can be further divided into subdomains 1 and 2, and the larger domain consists of subdomains 3 and 4. Proteolysis studies and fluorescence resonance energy transfer assays reveal shifts in the position of subdomain 2 in ATP vs. ADP monomeric actin (9-11). Consistent with this finding, three-dimensional reconstructions of electron micrographs of actin filaments revealed a disordering of subdomain 2 in ADP compared with ADPBeF actin filaments (12, 13).To study the mechanism by which actin uses the energy of ATP hydrolysis to control filament dynamics, we introduced mutations into the ATP binding pocket of the yeast actin gene.Here we discuss the study of actin in which Val-159 is mutated to Asn (V159N). The amide group of V159 makes a hydrogen bond wi...

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supporting

confidence: 64%

A change in actin conformation associated with filament instability after P _i release

Belmont

Orlova

Drubin

1999

Proc. Natl. Acad. Sci. U.S.A.

155

147

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“…This hinge and its surrounding were suggested to transmit conformational changes from the C-terminal segment to the nucleotide cleft [11]. On the other hand, the conformational relationship of the nucleotide cleft with the DNase I-binding loop [4,11,16] as well as with the 18~9 segment [17] has been reported. Reciprocal communication from the C-terminus to the DNase I binding loop also seems possible as in C-terminal truncated actin cleavage with subtilisin slows down [18].…”

Section: Discussionmentioning

confidence: 99%

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

et al. 1996

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Alteration of the actin polypeptide chain within the DNnse I-binding loop by cleavage with E. coli A2 protease or suhtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374. Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can he energy transfer donors. It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin. These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer. We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling.

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“…In addition to and independent of the structural and modeling data, there is considerable biochemical evidence that the nature of the bound nucleotide affects the conformation of the D-loop. For example, the intensity of fluorescence of a fluorescence-labeled Gln-41 is greater for G-ATP-actin than for G-ADP-actin (35,36), and the D-loop of G-ADP-actin is less susceptible to proteolytic cleavage than the D-loop of G-ATP-actin (29,37).…”

Section: Properties Of Purified Mutantmentioning

confidence: 99%

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

Liu

Shu

Hong

et al. 2010

Journal of Biological Chemistry

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All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either

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Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41

Cited by 59 publications

References 32 publications

A change in actin conformation associated with filament instability after P _i release

A change in actin conformation associated with filament instability after P _i release

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

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Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41

Cited by 59 publications

References 32 publications

A change in actin conformation associated with filament instability after P i release

A change in actin conformation associated with filament instability after P i release

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

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A change in actin conformation associated with filament instability after P _i release

A change in actin conformation associated with filament instability after P _i release