Ordered two-dimensional arrays such as S-layers 1 , 2 and designed analogues 3 – 5 have intrigued bioengineers, 6 , 7 but with the exception of a single lattice formed with flexible linkers, 8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages. 9 – 12 Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building-blocks, and use it to design a p6m lattice. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly crystalline micrometer-scale arrays nearly identical (based on TEM and SAXS) to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces which we demonstrate can drive extensive receptor clustering, downstream protein recruitment, and signaling. Using AFM on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and thus that our material can impose order onto fundamentally disordered substrates like cell membranes. In sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards a synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems.

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

The kinesin MCAK binds to end-binding proteins and antagonizes centrosome separation and promotes robust kinetochore attachments to spindle microtubules.

Asymmetric multiprotein complexes that undergo subunit exchange play central roles in biology but present a challenge for design because the components must not only contain interfaces that enable reversible association but also be stable and well behaved in isolation. We use implicit negative design to generate β sheet–mediated heterodimers that can be assembled into a wide variety of complexes. The designs are stable, folded, and soluble in isolation and rapidly assemble upon mixing, and crystal structures are close to the computational models. We construct linearly arranged hetero-oligomers with up to six different components, branched hetero-oligomers, closed C4-symmetric two-component rings, and hetero-oligomers assembled on a cyclic homo-oligomeric central hub and demonstrate that such complexes can readily reconfigure through subunit exchange. Our approach provides a general route to designing asymmetric reconfigurable protein systems.