title: Structure of the AvrBs3-DNA complex provides new insights into the initial thymine-recognition mechanism.
abstract: Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein-DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152-895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish Cancer Research Centre (CNIO), Melchor Fernandez Almagro, 28029 Madrid, Spain.
authors.forename: Stefano
authors.initials: S
authors.lastname: Stella
authors.forename: Rafael
authors.initials: R
authors.lastname: Molina
authors.forename: Igor
authors.initials: I
authors.lastname: Yefimenko
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: George
authors.initials: G
authors.lastname: Silva
authors.forename: Claudia
authors.initials: C
authors.lastname: Bertonati
authors.forename: Alexandre
authors.initials: A
authors.lastname: Juillerat
authors.forename: Phillippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D003460:Crystallization; D018360:Crystallography, X-Ray; D016373:DNA, Antisense / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004151:Dipeptides / Q000737:chemistry / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry*; D000071336:Transcription Activator-Like Effectors; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D016373:DNA, Antisense; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004151:Dipeptides; D009692:Nucleic Acid Heteroduplexes; D000071336:Transcription Activator-Like Effectors; C073876:avrBs3-2 protein, Xanthomonas campestris; D013941:Thymine
keywords: gene targeting; genetics; protein–DNA interaction