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. In addition, we discuss modifications to motor proteins that alter directionality, processivity, and cargo specificity, providing insights into their roles in cellular transport. Rapid advances in de novo protein design offer new tools to hijack natural cytoskeletal machinery and create synthetic elements for cellular architecture, including membraneless organelles and synthetic cytoskeletal tracks. This research promises to deepen our understanding of cellular organization, uncover regulatory mechanisms, and provide new proteins for therapeutic applications and synthetic cell development.

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. These different membrane morphologies likely reflect different patterns of nucleator self-organization. N-WASP phase separation has been successfully studied through biochemical reconstitutions, but how the WAVE complex self-organizes to instruct lamellipodia is unknown. Because WAVE complex self-organization has proven refractory to cell-free studies, we leverage in vivo biochemical approaches to investigate WAVE complex organization within its native cellular context. With single molecule tracking and molecular counting, we show that the WAVE complex forms highly regular multi-layered linear arrays at the plasma membrane that are reminiscent of a microtubule-like organization. Similar to the organization of microtubule protofilaments in a curved array, membrane curvature is both necessary and sufficient for formation of these WAVE complex linear arrays, though actin polymerization is not. This dependency on negative membrane curvature could explain both the templating of lamellipodia and their emergent behaviors, including barrier avoidance. Our data uncover the key biophysical properties of mesoscale WAVE complex patterning and highlight an integral relationship between NPF self-organization and cell morphogenesis.

De novo 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. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses α-helical barrel (αHB) peptide assemblies, which comprise 5 or more α helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, αHBs are analogous to "molecular flasks" made in supramolecular, polymer, and materials chemistry. Using Förster resonance energy transfer as a readout, we demonstrate that specific αHBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile red, in close proximity. In addition, two anthracene molecules can be accommodated within an αHB to promote anthracene photodimerization. However, not all ternary complexes are productive, either in energy transfer or photodimerization, illustrating the control that can be exerted by judicious choice and design of the αHB.

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. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) low-conductance states that correlate with variations in the designed amino-acid sequences and modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single channels change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic channels, as observed in natural barrel-stave peptide channels. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

Computational protein design is advancing rapidly. However, approaches and methods are needed to increase success rates and to elaborate designs. Here we describe the combination of rational and computational design to deliver three-helix bundle (3HB) peptide assemblies and single-chain proteins with control over topology and thermal stability. First, we garner sequence-to-structure relationships from antiparallel 3HBs in the Protein Data Bank. This gives core-packing rules, including layers of hydrogen-bonded polar residues, which are combined with surface-charge patterning to design complementary sequences for acidic (A), basic (B), and neutral (N) helices. By altering the design of the N helix, two sets of synthetic peptides are generated for clockwise and anticlockwise arrangements of the three-helix assemblies. Solution-phase characterisation shows that both ABN peptide mixtures form stable, heterotrimeric assemblies consistent with the targeted 'up-down-up' topologies. Next, AlphaFold2 models for both designs are used to seed computational designs of single-chain proteins where the helices are connected by loop building. Synthetic genes for these express in E. coli to yield soluble, monomeric, and thermally stable proteins. By systematically introducing additional polar layers within the core, the thermal stability of these proteins is varied without compromising the specificity of the helix-helix interactions. Chemical and thermal denaturation reveals comparable thermodynamic parameters to those of highly stable natural proteins. Four X-ray crystal structures confirm that the design models and AlphaFold2 predictions match to sub-Å accuracy.

Kinesin-1 is a dynamic heterotetrameric assembly of two heavy and two light chains (KHC and KLC) that mediates microtubule-based intracellular transport of many different cargoes. The complex adopts a compact, autoinhibited state that is activated by cargo-adaptor proteins containing specific short linear peptide motifs (SLiMs). These motifs interact with the tetratricopeptide repeat (TPR) domains of the KLCs. The mechanism coupling SLiM recognition to activation-associated conformational changes in the complex is unknown. Here we combine protein design, computational modelling, biophysical analysis, and electron microscopy to examine the structural and mechanistic consequences of SLiM binding to the KLC-TPR domain within the complete heterotetrameric holoenzyme. We show that coiled coil 1 (CC1) of the KHC docks KLC TPR domains in the autoinhibited complex, forming the “shoulder” feature observed in electron microscopy. Disrupting this interaction or binding an activating SLiM dislocates the TPR shoulder, freeing the motor domains and promoting transition between its closed, inactive, and open states. Therefore, cargo-mediated dislocation of the TPR shoulder serves as a key initial step in kinesin-1 activation, allosterically linking cargo binding to motor dynamics. ### Competing Interest Statement The authors have declared no competing interest.

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. Hot-spot residues from an MCL-1 binding peptide (NOXA-B) are grafted onto the outer surfaces of homo- and heterodimeric coiled-coil peptides to obtain inhibitors with mid-nM potency and selectivity over BCL-xL. Binding of homodimeric coiled coils to MCL-1 is positively cooperative, resulting in stabilization of both partners. Homodimeric coiled coils support the binding of two copies of the target protein. Modification of the coiled-coil sequence to favor assembly of higher-order scaffolds (trimer and tetramer) negatively impacts inhibitory potency, with AlphaFold2 modeling and biophysical data indicating a complex interplay between coiled-coil oligomerization and target binding. Together, these data establish dimeric coiled coils as the most promising of such scaffolds to develop inhibitors of α-helix-mediated PPIs.

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Combining molecular modelling, machine-learned models and an increasingly detailed understanding of protein chemistry and physics, computational protein design and human expertise have been able to produce new protein structures, assemblies and functions that do not exist in nature. Currently, generative deep-learning-based methods, which exploit large databases of protein sequences and structures, are revolutionizing the field, leading to new capabilities, improved reliability and democratized access in protein design. This Primer provides an introduction to the main approaches in computational protein design, covering both physics-based and machine-learning-based tools. It aims to be accessible to biological, physical and computer scientists alike. Emphasis is placed on understanding the practical challenges arising from limitations in our fundamental understanding of protein structure and function and on recent developments and new ideas that may help transcend these. Computational protein design uses information on the constraints of the biological and physical properties of proteins for protein engineering and de novo protein design. In this Primer, Albanese et al. give an overview of the guiding principles of computational protein design and its considerations, methods and applications and conclude by discussing the future of the technique in the context of rapidly advancing computational tools.

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. Here we introduce the concept of 'interface flexibility' and demonstrate its critical importance in the nucleation and growth of supramolecular networks. As a model system, we use trisymmetric DNA-based macromonomers, which organize into hexagonal networks through weak pi-pi interactions at their tips. The directional nature and low spatial tolerance of pi-pi interactions mean that small shifts in orientation have a large effect on effective valency. We show that too much interface flexibility disrupts network formation, regardless of affinity. Tuning the interface flexibility greatly expands the available design space for synthetic supramolecular materials.

The field of protein design has changed dramatically over the last 40 years, with a range of methods developing from rational design to more recent data-driven approaches. While considerable insight could be gained from analysing designed proteins, there is no single resource that brings together all the relevant data, making it difficult to identify current challenges and opportunities within the field. Here we present the Protein Design Archive, a website and database of designed proteins. Using the database, we performed systematic analysis that reveals a rapid increase in the number and complexity of designs over time and uncovers biases in their amino-acid usage and secondary structure content. Finally, sequence- and structure-based analysis demonstrates the breadth and novelty of designs within the archive. The PDA will be a valuable resource for guiding development of protein design, helping the field reach its potential. The PDA is available freely and without registration at . ### Competing Interest Statement The authors have declared no competing interest. All data generated in this study is available on the PDA Database website, downloadable as JSON or CSV file, and on GitHub, at the following address: .

Driven by deep-learning approaches, computational protein design is advancing rapidly, and it is now possible to generate many de novo protein structures quickly and robustly. This sets new frontiers for the field, including designing proteins that bind small molecules tightly and specifically, and understanding the non-covalent interactions that underpin such designs to make binding predictable and tunable. Here we address these challenges with a rapid physics-based computational method to generate isosteric and chemically complementary binding pockets for small-molecule targets in de novo designed proteins. We test this experimentally by constructing and characterizing binding proteins for several synthetic and natural chromophores. By evaluating only single-digit numbers of designs, the pipeline delivers stable proteins with pre-organized binding sites confirmed by X-ray crystallography, which bind the targets selectively with micromolar affinities or better. To illustrate the scope and applications of this approach, we incorporate distinct and coupled chromophore-binding sites in a two-domain de novo protein enabling controlled energy transfer between the two sites, and we develop a small de novo binding protein that can be used in live mammalian cells to visualize sub-cellular structures. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/X003027/1, BB/T008741/1 Engineering and Physical Sciences Research Council, EP/T012455/1 Leverhulme Trust, RGP-2021-049 Royal Society, URF\R\201007

De novo 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 de novo designed structures. Here, we explore one aspect of dynamics in a series of de novo coiled-coil peptide assemblies: namely, peptide exchange within and between different oligomers from dimers through to heptamers. First, we develop a fluorescence-based reporter assay for peptide exchange that is straightforward to implement, and, thus, would be useful to others examining similar systems. We apply this assay to explore both homotypic exchange within single species, and heterotypic exchange between coiled coils of different oligomeric states. For the former, we provide a detailed study for a dimeric coiled coil, CC-Di, finding a half-life for exchange of 4.2 +/- 0.3 minutes at a peptide concentration of 200 mu M. Interestingly, more broadly when assessing exchange across all of the oligomeric states, we find that some of the designs are faithful and only undergo homotypic strand exchange, whereas others are promiscuous and exchange to form unexpected hetero-oligomers. Finally, we develop two design strategies to improve the orthogonality of the different oligomers: (i) using alternate positioning of salt bridge interactions; and (ii) incorporating non-canonical repeats into the designed sequences. In so doing, we reconcile the promiscuity and deliver a set of faithful homo-oligomeric de novo coiled-coil peptides. Our findings have implications for the application of these and other coiled coils as modules in chemical and synthetic biology.

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.

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, A3B3 heterohexamer α-helical barrels. These designs are based on pairs of sequences with three heptad repeats (abcdefg), programmed with a = Leu, d = Ile, e = Ala, and g = Ser, and b = c = Glu to make the acidic (A) chains and b = c = Lys in the basic (B) chains. These design rules ensure that the desired oligomeric state and stoichiometry are readily achieved. However, controlling the orientation of neighboring helices (parallel or antiparallel) is less straightforward. Surprisingly, we find that assembly and helix orientation are sensitive to the length of the overhang between helices. To study this, cyclically permutated peptide sequences with three heptad repeats (the register) in the peptide sequences were analyzed. Peptides starting at g (g-register) form a parallel 6-helix barrel in solution and in an X-ray crystal structure, whereas the b- and c-register peptides form an antiparallel complex. In lieu of experimental X-ray structures for b- and c-register peptides, AlphaFold-Multimer is used to predict atomistic models. However, considerably more sampling than the default value is required to match the models and the experimental data, as many confidently predicted and plausible models are generated with incorrect helix orientations. This work reveals the previously unknown influence of the heptad register on helical overhang and the orientation of α-helical coiled-coil peptides and provides insights for the modeling of oligopeptide coiled-coil complexes with AlphaFold.

In targeted protein degradation (TPD) the cell's quality control machinery is co-opted to degrade proteins of interest. Currently TPD is limited by the availability of small-molecule binders for targets and the many potential human E3 ubiquitin ligases. Consequently, other approaches are needed to tackle new targets and to exploit degradation pathways fully. The natural binding epitopes for E3s and other degradation machineries are often short linear peptide motifs, which offer routes to new biologics-based degraders. Here, targeting the anti-apoptotic BCL-xL and initiation of apoptosis, we show that several protein-degradation pathways - ubiquitination, direct proteasome, autophagy-lysosome recruitment - can be leveraged by integrating target-binding and degradation motifs into single-polypeptide constructs. This exploits versatile and varied de novo coiled-coil scaffolds as adaptable molecular glues by presenting motifs multivalently and in diverse arrangements. The constructs are small (~80 residues) and provide a plug-and-play platform to map and optimise target-degradation space using rational design. We call this Coiled Coils for induced Proximity (CoCoProx) and propose its use for targeted degradation and other induced-proximity reactions. ### Competing Interest Statement The authors have declared no competing interest. BBSRC, BB/V008412/1, BB/V008412/2, BB/Y007816/1, BB/V003577/1, BB/V003577/2 Cancer Research UK, https://ror.org/054225q67, A29256

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

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. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane α-helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACTDe novoprotein 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. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses α-helical barrel (αHB) peptide assemblies, which comprise 5 or more α-helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, αHBs are analogous to ‘molecular flasks’ made in supramolecular, polymer, and materials chemistry. Using Förster resonance energy transfer as a readout, we demonstrate that specific αHBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an αHB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the αHB.

Computational protein design is advancing rapidly. Here we describe efficient routes to two families of α-helical-barrel proteins with central channels that bind small molecules. The designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides. Adjacent helices are connected using computational 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 computationally. Throughout these pipelines, residues that define open states of the barrels are maintained. This minimises sequence sampling and accelerates routes to successful designs. For each of 6 targets, just 2 – 6 synthetic genes are made for expression in E. coli . On average, 80% express to give soluble monomeric proteins that are characterized fully, including high-resolution structures for most targets that match the seed structures and design models with high accuracy.