Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

Sawaya, M.R.; Kudryashov, Dmitri S.; Pashkov, I.; Adisetiyo, Helty; Reisler, Emil; Yeates, Todd O.

doi:10.1107/s0907444908003351

Cited by 18 publications

(18 citation statements)

References 60 publications

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“…This similarity has been also observed in other crystal forms23; 24, highlighting the likelihood that these contacts will be recapitulated in the F-actin filament. What has not yet been seen is the full rotation of the monomers that will likely need to occur to generate F-actin.…”

Section: Discussionsupporting

confidence: 76%

Crystal Structures of Monomeric Actin Bound to Cytochalasin D

Nair

Joel

Wan

et al. 2008

Journal of Molecular Biology

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The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin has not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD, one obtained by soaking preformed actin crystals with CD, and the other by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play an equally important role in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft, and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site, and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. A rotation of ~6° of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure, instead of being disordered as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

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Section: Discussionsupporting

confidence: 76%

Crystal Structures of Monomeric Actin Bound to Cytochalasin D

Nair

Joel

Wan

et al. 2008

Journal of Molecular Biology

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“…1) (Oda et al 2007). These axial interactions are stronger, as evidenced by the observation of only subdomains 3 and 4 contacts in longitudinal dimer crystal structures (for a discussion of the longitudinal contacts observed in these structures, see (Sawaya et al 2008)). Radial interactions involve those interactions at far radius in F-actin and interactions between the subunits of F-actin.…”

Section: F-actin Interactions: Nucleotide-dependent Changesmentioning

confidence: 87%

“…Growing evidence suggests that different modes of intersubunit interactions are possible in F-actin, resulting in different structural states (Galkin et al 2001;Galkin et al 2002;Schmid et al 2004). Recent crystal structures have provided a partial picture of the long-pitch interactions that might be seen in Factin (Klenchin et al 2006;Kudryashov et al 2005;Nair et al 2008;Sawaya et al 2008). These interactions may be important for the mechanism of actin filament growth, since the first step in F-actin growth is thought to be a long-pitch dimer (Sept and McCammon 2001).…”

Section: Introductionmentioning

confidence: 95%

Actin polymerization is controlled by residue size at position 204

Yates

Loncar

Dawson

2009

Biochem. Cell Biol.

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Previous work has shown that purified double mutant A204C/C374A yeast actin is polymerization-deficient in vitro under physiological concentrations. To understand the importance of the 204 residue in subdomain 4, a series of actin proteins with a single mutation at this position were created with Cys-374 retained. Only yeast expressing A204G-, A204S-, or A204C-actin were viable. The A204G and A204S strains were sensitive to cold temperature and hyperosmolarity, whereas the A204C strain showed more profound effects on growth under these conditions. Cells expressing A204C-actin exhibited anomalies previously observed for A204C/C374A actin, including abnormal actin structures. A204G- and A204S-actin proteins had 12- and 13-fold increased critical concentrations, respectively, relative to wild-type. Only at very high concentrations could A204C actin polymerize when ATP was bound; when hydrolyzed, the ADP-containing A204C filaments depolymerized, demonstrating a profound difference in critical concentration between ATP and ADP states with A204C actin. A correlation between size of the residue substituted at position 204 and energy minimization of actin filament models was observed. We propose that the region surrounding residue 204 is involved in interactions that change depending on the phosphorylation state of the bound nucleotide that might reflect different conformations of F-actin subunits.

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“…However, recent crystal structures of crosslinked longitudinal dimers are promising (Kudryashov et al 2005;Sawaya et al 2008). While these dimers do not possess the helical twist seen in F-actin, the structures do suggest some longitudinal contacts between actin subunits that may be present in F-actin.…”

Section: Introductionmentioning

confidence: 93%

“…5C and 5D. (Kudryashov et al 2005;Sawaya et al 2008). We mixed D288C and A204C mutant ACT1 proteins (both in a C374A background and termed DC and AC actin, respectively) in the absence of reducing agent and monitored polymerization by light scattering (Fig.…”

Section: Examination Of F-actin Structure By Crosslinkingmentioning

confidence: 99%

Cysteine engineering of actin self-assembly interfaces

Pengelly

Loncar

Perieteanu

et al. 2009

Biochem. Cell Biol.

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The Holmes model of filamentous actin (F-actin) and recent structural studies suggest specific atomic interactions between F-actin subunits. We tested these interactions through a cysteine-engineering approach with the goal of inhibiting filament formation by introducing chemical groups at sites important for polymerization. We substituted surface amino acids on the actin molecule with cysteine residues and tested the effect of producing these actin mutant proteins in a yeast expression system. The intrinsic folding and polymerization characteristics of the cysteine-engineered actin proteins were measured. The effect of chemical modification of the introduced cysteine residues on the polymerization of the actin mutant proteins was also examined. Modification of cysteine residues with large hydrophobic reagents resulted in polymerization inhibition. We examined the finding that the D288C actin protein does not polymerize under oxidizing conditions and forms protein aggregates when magnesium and EGTA are present. Chemical crosslinking experiments revealed the presence of a lower dimer when only D288C actin was present. When both D288C and A204C actin were present, crosslinking experiments support the proximity of Asp288 on the barbed end of one subunit to Ala204 on the pointed end of a neighboring subunit in the Holmes model of F-actin.

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Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

Cited by 18 publications

References 60 publications

Crystal Structures of Monomeric Actin Bound to Cytochalasin D

Crystal Structures of Monomeric Actin Bound to Cytochalasin D

Actin polymerization is controlled by residue size at position 204

Cysteine engineering of actin self-assembly interfaces

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