Controlling cell architecture with protein design.

Cells depend on a complex and precisely regulated subcellular organization, largely driven by the cytoskeleton and motor proteins that control intracellular transport. This review explores innovative strategies to manipulate cellular architecture using targeted protein design and engineering of cytoskeletal elements and molecular motors. We highlight advances in inducible dimerization techniques, which enable precise control over cytoskeletal dynamics through light- and small-molecule-sensitive domains.

WAVE complex forms linear arrays at negative membrane curvature to instruct lamellipodia formation.

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.

Grafted Coiled-Coil Peptides as Multivalent Scaffolds for Protein Recognition.

Self-assembled peptides are promising templates for the design of inhibitors of protein-protein interactions (PPIs) because they can be endowed with affinity- and selectivity-defining amino acids alongside favorable physicochemical properties such as solubility and stability. Here, we describe a tunable coiled-coil scaffold and its interaction with MCL-1, an α-helix-binding antiapoptotic protein and important target in oncology. We explore the role of oligomerization, multivalency, and cooperativity in PPI inhibition.

De Novo Design of Parallel and Antiparallel AB Heterohexameric α-Helical Barrels.

The de novo design of α-helical coiled-coil peptides is advanced. Using established sequence-to-structure relationships, it is possible to generate various coiled-coil assemblies with predictable numbers and orientations of helices. Here, we target new assemblies, namely, AB heterohexamer α-helical barrels.

Rational Design Principles for α-Helical Peptide Barrels with Dynamic Conductive Channels.

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane α-helical barrels with conductive channels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles.

Interface flexibility controls the nucleation and growth of supramolecular networks.

Supramolecular networks are abundantly present in nature and, like crystalline materials, often develop from an initial nucleation site, followed by growth based on directional interactions between components. Traditionally, the binding strength and directionality of interactions is thought to dictate nucleation and crystal growth, whereas structural flexibility favours defects. Usually, macromonomers present multiple binding sites with relative intramolecular flexibility, but the effects of such flexibility on regulating network formation have been given little attention.

Confinement and Catalysis within Designed Peptide Barrels.

protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities.

Exchange, promiscuity, and orthogonality in designed coiled-coil peptide assemblies.

protein design is delivering new peptide and protein structures at a rapid pace. Many of these synthetic polypeptides form well-defined and hyperthermal-stable structures. Generally, however, less is known about the dynamic properties of the designed structures.

The WAVE complex forms linear arrays at negative membrane curvature to instruct lamellipodia formation.

Cells generate a wide range of actin-based membrane protrusions for various cell behaviors. These protrusions are organized by different actin nucleation promoting factors. For example, N-WASP controls finger-like filopodia, whereas the WAVE complex controls sheet-like lamellipodia.

Understanding β-strand mediated protein-protein interactions: tuning binding behaviour of intrinsically disordered sequences by backbone modification.

A significant challenge in chemical biology is to understand and modulate protein-protein interactions (PPIs). Given that many PPIs involve a folded protein domain and a peptide sequence that is intrinsically disordered in isolation, peptides represent powerful tools to understand PPIs. Using the interaction between small ubiquitin-like modifier (SUMO) and SUMO-interacting motifs (SIMs), here we show that -methylation of the peptide backbone can effectively restrict accessible peptide conformations, predisposing them for protein recognition.

Rationally seeded computational protein design of ɑ-helical barrels.

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.

A de novo designed coiled coil-based switch regulates the microtubule motor kinesin-1.

Many enzymes are allosterically regulated via conformational change; however, our ability to manipulate these structural changes and control function is limited. Here we install a conformational switch for allosteric activation into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed autoinhibited states.

Maturation and Conformational Switching of a Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation.

An atlas of protein homo-oligomerization across domains of life.

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life.

CC : A searchable database of validated coiled coils in PDB structures and AlphaFold2 models.

α-Helical coiled coils are common tertiary and quaternary elements of protein structure. In coiled coils, two or more α helices wrap around each other to form bundles. This apparently simple structural motif can generate many architectures and topologies.

Assembling membraneless organelles from de novo designed proteins.

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological processes and augment natural systems. The bottom-up design of self-assembled objects such as microcompartments and membraneless organelles is one such challenge.

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs.

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable antiparallel and parallel coiled-coil heterotetramers, , AB systems, to afford control in downstream applications.

Evaluation and deployment of isotype-specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults.

Background: Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection, but there is limited evidence on the utility of salivary antibody testing for community surveillance.

Methods: We established 6 ELISAs detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. We evaluated diagnostic performance, and using paired saliva and serum samples, correlated mucosal and systemic antibody responses.

Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils.

Protein science is being transformed by powerful computational methods for structure prediction and design: AlphaFold2 can predict many natural protein structures from sequence, and other AI methods are enabling the de novo design of new structures. This raises a question: how much do we understand the underlying sequence-to-structure/function relationships being captured by these methods? This perspective presents our current understanding of one class of protein assembly, the α-helical coiled coils. At first sight, these are straightforward: sequence repeats of hydrophobic (h) and polar (p) residues, (hpphppp), direct the folding and assembly of amphipathic α helices into bundles.