Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

Kabsch, Wolfgang; Mannherz, Hans Georg; Suck, Dietrich

doi:10.1002/j.1460-2075.1985.tb03900.x

Cited by 97 publications

(48 citation statements)

References 25 publications

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“…The shape ofthe scattering curve at larger values ofS shows that the actin molecules are not simple spheres but rather are asymmetric in shape. The calculated scattering pattern for a simple two-sphere model having approximate dimensions of actin in the DNase-actin crystal (35), yields good agreement with the experimentally observed pattern (unpublished results). Actin is filamentous in high ionic strength buffer.…”

Section: Methodssupporting

confidence: 71%

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Matsudaira¹,

Bordas²,

Koch³

1987

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

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Synchrotron x-ray diffraction was used to identify the oligomers that formed during the earliest stages of actin polymerization. Solution diffraction patterns from Gactin (monomer) and from F-actin (polymer) contain information about the size and shape of actin monomers and the length, width, and subunit organization of filaments. Comparison of patterns collected during polymerization reveals an increase in scatter at spacings >9.0 nm; formation of scattering bands at 5.4, 4.9, and 3.4 nm; formation of a scattering minimum at 6.5 nm; and the presence of an isosbestic point at 9.0 nm. These scattering bands arise from the formation of, and organization of subunits in, framents. At various actin concentrations (0.37-5 mg/ml), the change in scatter in these regions follows simple exponential kinetics with no detectable lag. Our analysis of the x-ray patterns shows that by 0.4 sec after mixing, most of the actin has formed dimers, which then rapidly incorporate into oligomers. At 4 mg/ml the early oligomers increase in length to >30.0 nm within 10 sec. These results suggest that under our conditions actin molecules condense into filaments without the rate-limiting formation of nuclei.Studies on the mechanism of actin polymerization in vitro have influenced our ideas on the regulation of actin function in vivo. Central to these studies, and based partly on the kinetics of helical polymerization, is the proposal by Oosawa and colleagues (1-3) that actin polymerization is a nucleation-condensation process. During the initial phase of assembly, three to four actin monomers cooperatively associate into a short oligomer that nucleates rapid filament elongation. Nucleation is the rate-limiting step in assembly and is detected in kinetic studies as a lag in the time course of polymerization. The existence and size of nuclei is deduced from modeling the kinetics of the elongation phase, assuming that nucleation is an obligatory step in the reaction, and from the presence of a critical concentration for assembly. Detailed analysis using sophisticated computer programs have modified the model for actin assembly to include (i) a first-order Mg2" activation step, (ii) nuclei composed of actin trimers, (iii) different rates of assembly at the ends of the filament, and (iv) enhanced nucleation by spontaneous filament breakage. Although a variety of methods, including viscometry (4,5), flow birefringence (6), light scattering (7-11), and fluorimetry (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21), have provided indirect evidence that nucleation is an important feature of actin assembly in vitro, no structural study has directly identified the existence or the structure of the nucleating species.Time-resolved x-ray diffraction using high-intensity synchrotron radiation sources has proved a powerful technique for describing the structure and assembly of proteins into multisubunit complexes. Studies on microtubule and collagen fiber assembly have shown that time-resolved x-ray scattering methods can identify the structures forme...

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Section: Methodssupporting

confidence: 71%

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Matsudaira¹,

Bordas²,

Koch³

1987

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

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“…Modification of Tyr-53 may sterically block phalloidin binding. Tyr-53 is located at the loop region where DNase I binds [lo] and the region is at some distance from the cleft region where phalloidin binds [6] according to the X-ray crystallography model [8]. However, the size of the phalloidin molecule is sufficient for the diazonium-tetrazole modification sterically to block actin binding.…”

Section: Discussionmentioning

confidence: 99%

Interaction of phalloidin with chemically modified actin

Miki

Barden

Remedios

et al. 1987

European Journal of Biochemistry

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Modification of Tyr-69 with tetranitromethane impairs the polymerizability of actin in accordance with the previous report [Lehrer, S. S. and Elzinga, M. (1972) Fed. Proc. 31, 5021. Phalloidin induces this chemically modified actin to form the same characteristic helical thread-like structure as normal F-actin. The filaments bind myosin heads and activate the myosin ATPase activity as effectively as normal F-actin. When a dansyl group is introduced at the same point [Chantler, P. D. and Gratzer, W. B. (1975) Eur. J. Biochem. 60,67-721, phalloidin still induces the polymerization. The filaments bind myosin heads and activate the myosin ATPase activity. These results indicate that Tyr-69 is not directly involved in either an actin-actin binding site or the myosin binding site on actin. Moreover, the results suggest that phalloidin binds to actin monomer in the presence of salt and its binding induces a conformational change in actin which is essential for polymerization, or that actin monomer fluctuates between in unpolymerizable and polymerizable form while phalloidin binds to actin only in the polymerizable form and its binding locks the conformation which causes the irreversible polymerization of actin.Modification of Tyr-53 with 5-diazonium-(1H)tetrazole blocks actin polymerization [Bender, N., Fasold, H., Kenmoku, A., Middelhoff, G. and Volk, K. E. (1976) Eur. J . Biochem. 64,215 -2181. Phalloidin is unable to induce the polymerization of this modified actin nor does it bind to it. Phalloidin does not induce the polymerization of the trypsin-digested actin core. These results indicate that the site at which phalloidin binds is involved in polymerization and the probable conformational change involved in polymerization may be modulated through this site.Phalloidin binds tightly to F-actin but not to G-actin in the absence of salt [l, 21 and stabilizes the filamentous structure of actin against depolymerizing conditions or agents such as heat denaturation and proteolytic degradation [3 -51. Vandekerckhove et al. [6] reacted actin with two types of phalloidin derivatives, each having different chemical targets. They concluded that the phalloidin binding site is located in the cleft between the two domains of the actin monomer and binds to F-actin around residues 117-119 and 355, though it is not yet clear whether these residues belong to the same monomer. Recently it has also been demonstrated from fluorescence resonance energy transfer measurements that the phalloidin binding site lies within 1.0 nm of the nucleotide binding site [7], while the nucleotide binding site is located in the cleft region by X-ray crystallography at 0.45-nm resolution Bovine pancreatic DNase I depolymerizes F-actin and forms a 1 : 1 complex with actin monomers [9]. It binds to a loop region in the small domain of actin [S] and is chemically cross-linked to the residues 50, 53, 61, 68 and/or 69 [lo]. Selective chemical modification of Tyr-53 with 5-diazonium-(1H)tetrazole [ll], Lys-61 with fluorescein-5-isothiocyanate (FITC) [12] o...

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“…Mayer et al (1984) demonstrated that in adult rat hearts, the ca-skeletal actin gene is DNase I sensitive whereas this is not the case in non-muscle cells. Moreover, there is already a low level of mRNA derived from this gene in normal adult rodent hearts Mayer et al, 1984 (Kabsch et al, 1985). The mechanical force of muscular contraction is generated at the interface between myosin heavy chain and actin in the sarcomere.…”

Section: Discussionmentioning

confidence: 99%

A 5′ duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

Garner¹,

Minty²,

Alonso³

et al. 1986

The EMBO Journal

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We describe the structure and transcriptional activity of the 5′ portion of the alpha‐cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha‐cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha‐skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.

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Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

Cited by 97 publications

References 25 publications

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Synchrotron x-ray diffraction studies of actin structure during polymerization.

Interaction of phalloidin with chemically modified actin

A 5′ duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

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