Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering

Abstract

In recent years, new protein engineering methods have produced more than a dozen symmetric, self‐assembling protein cages whose structures have been validated to match their design models with near‐atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X‐ray crystallography of designed protein oligomers that form two‐component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen‐bonding networks over exclusively hydrophobic interactions to stabilize the designed protein–protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen‐bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I213 is generated by an off‐target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.

Connectability of protein cages

“…In such approaches, a serious concern is poor production yield of target structures due to multiple assembly states or random protein aggregations, oen observed in articial protein cage formation. 32,35,73 Appropriate selection of site-specic bond formation chemistry as well as of building blocks based on a precise assembly prediction will be important for obtaining monodisperse protein nanoassemblies with desired morphologies and functions.…”

“…31 This symmetry-based approach has resulted in an impressive library of protein architectures including two-component cages with tetrahedral and icosahedral symmetries. [31][32][33][34][35] Cage-like structures can be constructed using protein building blocks that do not naturally form cages. This strategy was rst devised by the Yeates group (Fig.…”

A fragment-based protein interface design algorithm for symmetric assemblies

“…The dataset for the design recapitulation experiments was generated by selecting all successfully designed two-component tetrahedral, octahedral and icosahedral designs from previously published work [4][5][6]12,14,49] ; these cases met the criteria of agreement between the model and an experimentally determined atomic model. Failed designs (e.g.…”

“…When such precision can be achieved by design, diverse protein-based materials with tailored spatial and biochemical properties can be produced. As examples, cubic and icosahedral protein cages [2][3][4][5][6][7], as well as extended protein arrays [8][9][10][11], are finding wide ranging uses as biotherapeutics (e.g. for vaccines) [12][13][14], as scaffolds for enzyme organization or atomic imaging [15][16][17][18], and as nanoscale containers for molecular encapsulation and delivery [19,20].…”

“…Subsequent amino acid sequence design and additional filtering steps were required to identify interfaces that might exhibit native-like properties. Newer protocols have shown the value of considering known residue pair interactions during docking [22] and prioritizing interfacial hydrogen bonding during sequence design [6,25,29].…”