Figure 1.
TAL topology and RVD-to-DNA code.
(A) Sequence of a TAL protein: type III secretion system tag (T3SS-tag, violet), tandem-repeat domain (blue), nuclear localization signal (NLS, yellow) and acidic transcriptional activation domain (AD, green). The amino acid sequence of a single repeat is shown, highlighting the RVD region (X12 and X13). The secondary structure is reported underneath; the kink induced by P27 is represented as a break in the α2 rod. (B) Natural occurrence of the known RVDs is reported together with the targeted DNA base to highlight RVD selectivity [4]. While some RVDs target only a single base (e.g. HD and ND), others have shared affinities (e.g. NN and N*). (C) Representative structure of one repeat as extracted from the 3V6T structure; relevant molecular information is highlighted.
Figure 2.
(A) Top view of bound TAL along the DNA axis showing the N-terminus (structure from 3UGM). (B) Side view of bound TAL displayed from the N-terminus (bottom) to the C-terminus (top) (structure from 3UGM). (C) TAL without DNA and the RVDs explicitly depicted (laying on the inner-side of the super-helix). The orange line represents the DNA axis. The protein is orientated from N-terminus (bottom) to C-terminus (top). A larger pitch compared to the bound structure is clearly observable (structure from 3V6P). Protein: grey; DNA: orange.
Table 1.
Simulation details for each system.
Figure 3.
Structural and energetic features of TAL-DNA interaction.
(A) Mean residue fluctuation (RMSD) computed for the DNA-bound and apo states of the 11.5-repeats TAL system (TAL[11.5]/P1 and TAL[11.5]/P1-apo); averages are performed over all the repeats; bars represent standard deviations. The same trend is observed for all simulated systems (cf. Figure S5). (B) Contribution to the total DNA-binding energy from different sections of TAL subdivided by type and calculated on the DNA-bound 22.5-repeat TAL system (TAL[22.5]/P1) using MM/GBSA (Number of residues contributing to each type: G13 = 6, N13 = 2, I13 = 7, D13 = 5, G14/K16/Q17 = 20, others = 600, N-terminus = 97). Repeats containing a deletion at position X13 have been excluded from the statistics. (C) Per repeat mean energy contribution to the total DNA-binding energy; averages are performed over all the repeats of the DNA-bound 22.5-repeat TAL system (TAL[22.5]/P1). Repeats containing a deletion at X13 position have been excluded from the statistics. The complete binding energy profile is reported in Figure S9.
Figure 4.
Schematic representation of the TAL-DNA interface.
(A) Cartoon representation of one single repeat interacting with DNA. The oxyanion clip (G14, K16 and Q17) interacts with the phosphate group of the (i-1)th base, thus fixing the position of the X13 side-chain with respect to the ith base and freezing its structural fluctuation. Dashed circles indicate the interaction radii of different X13 residues, sorted by side-chain size: the inner circle corresponds to G13/*13, while the others are represented by the outer circle. Only loci α, β and γ are sampled by the side-chains of X13, resulting in incomplete molecular differentiation. (B) Pharmacophore-like model for the nucleobases discussed in the text (left). Dots represent sites with variable properties across nucleobases; colours are used to highlight the characteristics of the substituents: green for pyridine-like (H-bond acceptor) nitrogen atoms, blue for pyrrole-like and amine (H-bond donor) nitrogen atoms, grey for methyl groups, and red for carbonyl oxygen atoms. On the right, relative molecular electrostatic potential (MEP) maps for the corresponding nucleobases are reported. Calculations were performed at the QM level on methyl-capped purines (N9) and pyrimidines (N1) (red = −5.0 kBT/e, blue = 5.0 kBT/e, isovalue 4.0 E−4).
Figure 5.
Protein-DNA interactions of potentially improved RVD loops for targeting guanine.
(A) Wild type (N12-N13) alone (grey) and superposed to: (B) N12-N13K mutant (yellow), (C) N13K-G14* mutant (green) and (D) N13*-G14K mutant (magenta).
Table 2.
Protein-DNA binding energies for modified targets of repeat 7 (N*) of the PthXo1 system (TAL[22.5]/P1).