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Different actin nucleation-promoting factors (NPFs) orchestrate different patterns of cell protrusions, likely reflecting their distinct patterns of self-organization. Here, we leveraged in vivo biochemical approaches to investigate how the WAVE complex instructs the formation of sheet-like lamellipodia. We show that the WAVE complex is a core constituent of a linear multilayered protein array at the plasma membrane, expected for an NPF that builds sheet-like actin-based protrusions. Negative membrane curvature is both necessary and sufficient for WAVE complex linear membrane association in the presence of upstream activators (Rac, Arf1/6, and PIP3) and the PRDs of both WAVE2 and Abi2, providing a potential mechanistic basis for templating of lamellipodia and their emergent behaviors, including barrier avoidance. Through computational modeling, we demonstrate that WAVE complex’s linear organization and preference for negative curvature both play important roles in robust lamellipodia formation. Our data reveal key features of mesoscale WAVE complex patterning and highlight an integral relation between NPF self-organization and cell morphogenesis. 

Abstract Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix–turn–helix–turn–helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression inEscherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy. 

The design of completely synthetic proteins from first principles— de novo protein design—is challenging. This is because, despite recent advances in computational protein–structure prediction and design, we do not understand fully the sequence-to-structure relationships for protein folding, assembly, and stabilization. Antiparallel 4-helix bundles are amongst the most studied scaffolds for de novo protein design. We set out to re-examine this target, and to determine clear sequence-to-structure relationships, or design rules, for the structure. Our aim was to determine a common and robust sequence background for designing multiple de novo 4-helix bundles. In turn, this could be used in chemical and synthetic biology to direct protein–protein interactions and as scaffolds for functional protein design. Our approach starts by analyzing known antiparallel 4-helix coiled-coil structures to deduce design rules. In terms of the heptad repeat, abcdefg — i.e. , the sequence signature of many helical bundles—the key features that we identify are: a = Leu, d = Ile, e = Ala, g = Gln, and the use of complementary charged residues at b and c. Next, we implement these rules in the rational design of synthetic peptides to form antiparallel homo- and heterotetramers. Finally, we use the sequence of the homotetramer to derive in one step a single-chain 4-helix-bundle protein for recombinant production in E. coli . All of the assembled designs are confirmed in aqueous solution using biophysical methods, and ultimately by determining high-resolution X-ray crystal structures. Our route from peptides to proteins provides an understanding of the role of each residue in each design.