Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery: Bridging Experiments and Computations - September 10-14, 2014, Istanbul, Turkey

Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Poster Session II

76-POS Board 29 Design of Target Specific Transcription Activator-like Effectors by Free Energy Perturbation Calculations Sofia Piepoli , Batu Erman, Canan Atilgan. Sabanci University - Istanbul, Orhanlı Istanbul, Turkey. Bacterial plant pathogens belonging to Xanthomonas genus infect cells by injecting Transcription Activator-Like effector (TALE) proteins to modify plant gene expression. DNA binding TALE proteins have been modified to generate site specific engineered TALENs (transcription activator-like nucleases). Crystal structure of the PthXo1 TALE protein bound to its DNA target reveals specificity of interactions between the two molecules at the sequence level. TALE DNA binding domains include ~17 repeats, each consisting of 33-34 conserved amino acids. Alignment of the sequence of the repeats shows perfect homology except for the amino acids at positions 12-13 (repeat variable diresidues-RVDs) which specifically bind to DNA bases according to the code: HD>C, NG>T, NI>A and NN>G in most cases, while there are some exceptions. Not all repeats have to perfectly contact DNA to bind and it is unclear how TALE pairs up with a target DNA sequence while avoiding off-target sites. To address this problem, we apply molecular dynamics-based tools to quantify specific binding stability, mimicking cellular conditions. We analyze selected point mutations introduced into the RVD region of the protein sequence and predict resulting free energy differences using detailed free- energy perturbation calculation. Identification of key residues for stability of binding at specific positions will allow computational design of TALEs that can be experimentally tested for binding affinity and off-target specificity. By systematically applying this method, we interpret the role of local interactions in contributing to the overall stability of the system. For example, we find that RVD mutations HD→HA and NI→NN results in ΔG=14.4 and -19.8 kcal/mol, respectively, as expected from concurrent statistical studies. We test the effect of the same mutation in different repeats and discuss the implication of our findings in the context of local structural arrangements and allosteric effects.

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