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

Werther, Rachel; Hallinan, J.P.; Lambert, Abigail R.; Havens, Kyle A.; Pogson, Mark; Jarjour, Jordan; Galizi, Roberto; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony; Stoddard, Barry

doi:10.1093/nar/gkx544

Cited by 13 publications

(13 citation statements)

References 57 publications

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“…Taken in context with the current data, our studies reaffirm findings that meganuclease DNA interactions comprise of direct and indirect readouts of DNA sequence and structure, and highlight how structural reorganization of an inter-hairpin loop can dramatically change substrate specificity. That similar reorganizations have been observed in other engineered meganucleases lends support to our data (30), while at the same time suggesting that extensive substrate profiling of engineered variants may reveal changes in substrate specificity that were not rationally incorporated into those meganuclease scaffolds.…”

Section: Discussionsupporting

confidence: 89%

“…The residues we focused on here have been targeted by a number of directed-evolution studies to create variants with altered binding specificity at the +3, +4 and +5 positions of substrate (for example, (22–23,51–52)). Crystallographic studies of some of these engineered enzymes have noted similar changes to those we observed—restructuring of an inter hairpin loop, so-called role swapping where residues exchange DNA recognition for structural roles (or vice versa ) in response to different DNA substrates, and distortion of the DNA backbone (30). An under-appreciated consequence of meganuclease adaptability is the potential to indirectly modulate cleavage preference at the central 4 bases.…”

Section: Discussionsupporting

confidence: 61%

“…Using multiple sequence alignments of meganucleases and their cognate target sites, we identified and experimentally validated one residue–base combination that indirectly influences specificity of the I-OnuI meganuclease at the +2 position of the central 4 bases. Our findings show that substitutions in covarying residues can create new base-specific contacts as well as modulate cleavage preference, and suggest that some engineered meganucleases may have unanticipated cleavage preference that would only be revealed by extensive substrate profiling (30).…”

Section: Introductionmentioning

confidence: 84%

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Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Laforet

McMurrough

et al. 2019

Nucleic Acids Research

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Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 84%

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Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Laforet

McMurrough

et al. 2019

Nucleic Acids Research

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“…Crystallographic and biophysical analyses of five different extensively retargeted variants of a single meganuclease, that have been shown to function efficiently in ex vivo and in vivo applications, has been more recently reported ( 23 ). 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 (Figure 2D ).…”

Section: Homing Endonuclease (Meganuclease) Engineeringmentioning

confidence: 99%

Engineering altered protein–DNA recognition specificity

Bogdanove

Bohm

Miller

et al. 2018

Nucleic Acids Research

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Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein–protein interactions, modifying protein–DNA interactions is more difficult. This may be related to the structural features of protein–DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein–DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

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“…The eOnu_HIVInt_v1 structure was deposited into the RCSB protein database with the PDB ID 5V0Q and the eOnu_HIVInt_v2 structure with PDB ID 5T8D. The structure of 5T8D was recently described for the purpose of comparing the mechanism of shifting specificity between multiple redesigned variants of the same meganuclease scaffold (Werther et al, 2017). All of the engineering and activity studies and data in this article, including the structure of 5V0Q, are otherwise novel.…”

Section: Crystallization Structure Determination and Refinementmentioning

confidence: 99%

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses

Niyonzima

Lambert

Werther

et al. 2017

Protein Engineering, Design and Selection

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The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.

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

Cited by 13 publications

References 57 publications

Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Engineering altered protein–DNA recognition specificity

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses

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