Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity.

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites.

Design of functionalised circular tandem repeat proteins with longer repeat topologies and enhanced subunit contact surfaces.

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.

Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.

The redesign of a macromolecular binding interface and corresponding alteration of recognition specificity is a challenging endeavor that remains recalcitrant to computational approaches. This is particularly true for the redesign of DNA binding specificity, which is highly dependent upon bending, hydrogen bonds, electrostatic contacts, and the presence of solvent and counterions throughout the molecular interface. Thus, redesign of protein-DNA binding specificity generally requires iterative rounds of amino acid randomization coupled to selections. Here, we describe the importance of scaffold thermostability for protein engineering, coupled with a strategy that exploits the protein's specificity profile, to redesign the specificity of a pair of meganucleases toward three separate genomic targets. We determine and describe a series of changes in protein sequence, stability, structure, and activity that accumulate during the engineering process, culminating in fully retargeted endonucleases.

Engineering and functionalization of large circular tandem repeat protein nanoparticles.

Protein engineering has enabled the design of molecular scaffolds that display a wide variety of sizes, shapes, symmetries and subunit compositions. Symmetric protein-based nanoparticles that display multiple protein domains can exhibit enhanced functional properties due to increased avidity and improved solution behavior and stability. Here we describe the creation and characterization of a computationally designed circular tandem repeat protein (cTRP) composed of 24 identical repeated motifs, which can display a variety of functional protein domains (cargo) at defined positions around its periphery. We demonstrate that cTRP nanoparticles can self-assemble from smaller individual subunits, can be produced from prokaryotic and human expression platforms, can employ a variety of cargo attachment strategies and can be used for applications (such as T-cell culture and expansion) requiring high-avidity molecular interactions on the cell surface.

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.

LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.