129
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.
