TAL Effectors Specificity Stems from Negative Discrimination

Basile I M Wicky,Marco Stenta,Matteo Dal Peraro,Paolo Carloni

doi:10.1371/journal.pone.0080261

Basile I M Wicky, Marco Stenta + Show 2 more

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https://doi.org/10.1371/journal.pone.0080261

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Abstract

Transcription Activator-Like (TAL) effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD). The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks – allowing folding of TAL as a DNA-wrapping super-helix – the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools.

Highlights

TAL (Transcription Activator-Like) effectors are proteins secreted by phytopathogenic Gram-negative bacteria from the Xanthomonas genus [1], responsible for the infection of more than 200 different plant families, including many crops [2]
Based on the interpretation provided by the pharmacophorelike model, the large dominance of non-specific energetic contributions to TAL-DNA binding and the low fluctuation of the Repeat Variable Diresidue (RVD) loop – resulting in incomplete molecular recognition – we suggest that sequence specificity is not achieved through positive recognition of nucleobases but instead stems from negative discrimination, i.e. the match between base and RVD corresponds to the least bad option available when taking steric and electrostatic contributions into account
The dynamical study described in this report suggests that the composition of each RVD can be deconvoluted into its constituent parts and their contributions treated separately

Summary

Introduction

TAL (Transcription Activator-Like) effectors are proteins secreted by phytopathogenic Gram-negative bacteria from the Xanthomonas genus [1], responsible for the infection of more than 200 different plant families, including many crops [2]. TAL effectors are injected directly into the host plant cells via Type III Secretion System (T3SS) and, after localization to the nucleus, bind to DNA sequences, interfering with native cellular functions and supporting infection [1]. Endogenous TAL proteins are composed of an N-terminal translocation signal necessary for T3SS injection, a C-terminal nuclear localization signal (NLS) domain and an acidic activation domain (AD), both important eukaryotic motifs (Figure 1A) [2,3]. Polymorphism among repeats occurs predominantly at positions 4, 12, 13 and 32 (internal numbering), with positions 12 and 13 being by far the most variable, and termed Repeat Variable Diresidue (RVD) [2,4]

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