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High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.

Linkner J, Witte G, Stradal T, Curth U, Faix J - PLoS ONE (2011)

Bottom Line: We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes.Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography.Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.

ABSTRACT
The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.

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Proposed model for the assembly of the Scar/WAVE-complex.(A) Homotrimeric DdBrk1 maintains a dynamic pool of monomers, (B) which immediately associate with nascent Scar/WAVE and Abi proteins to form heterotrimeric subcomplexes. (C) Immediately thereafter, Scar/Abi/Brk1 heterotrimers are captured by the heterodimeric Nap/Pir platform to form the mature Scar/WAVE-complex (D).
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pone-0021327-g006: Proposed model for the assembly of the Scar/WAVE-complex.(A) Homotrimeric DdBrk1 maintains a dynamic pool of monomers, (B) which immediately associate with nascent Scar/WAVE and Abi proteins to form heterotrimeric subcomplexes. (C) Immediately thereafter, Scar/Abi/Brk1 heterotrimers are captured by the heterodimeric Nap/Pir platform to form the mature Scar/WAVE-complex (D).

Mentions: Based on these results and previous findings, in particular the observed incorporation of exogenously added oligomeric human Brk1 into the mature Scar/WAVE-complex in vivo [26], we suggest that a constant pool of Brk1 homotrimers allows for formation of a given number of monomers, which directly bind and stabilize de novo synthesized Scar/WAVE and Abi subunits (Fig. 6A,B). Since the activation of the Arp2/3-complex is a highly regulated process in vivo, we assume that heterotrimeric Scar/AbiA/DdBrk1-complexes immediately associate with the heterodimeric Nap/Pir platform to mask the VCA domain of Scar/WAVE proteins to prevent the unregulated activation of the Arp2/3-complex (Fig. 6C,D). This is consistent with previous observations showing that stability of Nap and Pir proteins is apparently not affected in the absence of Scar/WAVE, Abi or Brk1 proteins in vivo, and by the fact that heterodimeric Nap/Pir remains stable after coexpression in insect cells [21], [23], [31], [32], [36]. Notably, Brk1 forms the majority of the surface contacts between heterotrimeric HsBrk1/Abi2/WAVE1 and Nap/Pir platform within the trimeric coiled-coil region (Fig. 4A,B). We therefore propose that in the final assembly step, Brk1 serves as an adapter protein to stabilize and coordinate the assembly of the heterotrimer and the binding to the Pir/Nap platform to assemble the mature Scar/WAVE-complex.


High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.

Linkner J, Witte G, Stradal T, Curth U, Faix J - PLoS ONE (2011)

Proposed model for the assembly of the Scar/WAVE-complex.(A) Homotrimeric DdBrk1 maintains a dynamic pool of monomers, (B) which immediately associate with nascent Scar/WAVE and Abi proteins to form heterotrimeric subcomplexes. (C) Immediately thereafter, Scar/Abi/Brk1 heterotrimers are captured by the heterodimeric Nap/Pir platform to form the mature Scar/WAVE-complex (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3119050&req=5

pone-0021327-g006: Proposed model for the assembly of the Scar/WAVE-complex.(A) Homotrimeric DdBrk1 maintains a dynamic pool of monomers, (B) which immediately associate with nascent Scar/WAVE and Abi proteins to form heterotrimeric subcomplexes. (C) Immediately thereafter, Scar/Abi/Brk1 heterotrimers are captured by the heterodimeric Nap/Pir platform to form the mature Scar/WAVE-complex (D).
Mentions: Based on these results and previous findings, in particular the observed incorporation of exogenously added oligomeric human Brk1 into the mature Scar/WAVE-complex in vivo [26], we suggest that a constant pool of Brk1 homotrimers allows for formation of a given number of monomers, which directly bind and stabilize de novo synthesized Scar/WAVE and Abi subunits (Fig. 6A,B). Since the activation of the Arp2/3-complex is a highly regulated process in vivo, we assume that heterotrimeric Scar/AbiA/DdBrk1-complexes immediately associate with the heterodimeric Nap/Pir platform to mask the VCA domain of Scar/WAVE proteins to prevent the unregulated activation of the Arp2/3-complex (Fig. 6C,D). This is consistent with previous observations showing that stability of Nap and Pir proteins is apparently not affected in the absence of Scar/WAVE, Abi or Brk1 proteins in vivo, and by the fact that heterodimeric Nap/Pir remains stable after coexpression in insect cells [21], [23], [31], [32], [36]. Notably, Brk1 forms the majority of the surface contacts between heterotrimeric HsBrk1/Abi2/WAVE1 and Nap/Pir platform within the trimeric coiled-coil region (Fig. 4A,B). We therefore propose that in the final assembly step, Brk1 serves as an adapter protein to stabilize and coordinate the assembly of the heterotrimer and the binding to the Pir/Nap platform to assemble the mature Scar/WAVE-complex.

Bottom Line: We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes.Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography.Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.

ABSTRACT
The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.

Show MeSH
Related in: MedlinePlus