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.
Highlights
Cells harness the power of actin polymerization for the formation of protruding membrane sheets filled with a dense actin filament network at the leading edge referred to as lamellipodia to drive cell migration [1,2,3]
Brk1 is stable outside the functional Scar/WAVE-complex Brk1 is important for the stability of the Scar/WAVE subunit in different organisms [26,32,34,35], and was proposed to function as a precursor for Scar/WAVE-complex assembly in Hela S cells [26]
This assumption is further supported by the fact that, in addition to the Scar/WAVE-complex, an excess of Brk1 is found in the cytosol of HeLa S cells [22]
Summary
Cells harness the power of actin polymerization for the formation of protruding membrane sheets filled with a dense actin filament network at the leading edge referred to as lamellipodia (or pseudopodia in Dictyostelium) to drive cell migration [1,2,3]. Recent exciting work reporting on the structure of the human heteropentameric Scar/WAVE-complex, revealed details of its inactive state and how Rac binding could lead to the release of the masked VCA domain, activating the Scar/WAVE-complex [23]. In vertebrates, Brk forms homooligomers that remain stable as a free subcomplex in the absence of other Scar/WAVE-complex subunits [22,26]. This is remarkable, as the depletion of one subunit commonly leads to degradation of at least the Scar/WAVE and Abi proteins [26,27,28,29,30]. We present the high resolution structure and a biochemical analysis of DdBrk, which provides new insights on the question of how Brk may function as a precursor during the assembly of the mature Scar/WAVEcomplex
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