The structure of the T1414 dimer has been determined to 2.0 Å resolution. Each monomer is entirely α-helical and consists of two domains . Three helices (H1–H3) form the N-terminal DNA-binding domain with its characteristic HTH motif , and the remaining helices (H4-H10) form the C-terminal ligand-binding domain.
Figure 1 (A) Structure of T1414 dimer, with monomers shown in yellow and blue. α-helices are numbered H1 to H10. (B) Model of the T1414 dimer interacting with DNA (both macromolecules shown in backbone representation). (C) CLUSTALL25 sequence alignment (more ...)
search documented structural similarity among T1414 and six previously determined structures with Z-score more than 10, including (1) transcriptional regulator (1VI0), (2) transcriptional regulator QacR (1JTY),4
(3) putative transcriptional regulator YfiR (1RKT), (4) TetR family repressor MarR (1T56),13
(5) putative transcriptional repressor (TetR/Accr family) (1T33), and (6) Gamma-Butyrolactone receptor or ARPA-like protein (1UI5).24
1RKT and 1T33 were determined by structural genomics programs and were not present in the PDB at the time of target selection. Despite very low sequence identity (< 16%) between T1414 and each of these six structurally related proteins, they are remarkably similar to T1414 [root mean square deviations (RMSD) for common Cα atoms less than 2.5 Å]. The sequence alignment (CLUSTALW)25
of these structures with T1414 shows that considerable similarity exists within ~ 65 N-terminal amino acids containing the putative HTH motif. Particularly Ala15, Phe47, and Glu52 are strictly conserved  and the other region shows a very poor sequence similarity [region not shown in figure, ]. Not surprisingly, there is closer similarity among the N-terminal DNA-binding domains (15%–42% identity), and more considerable divergence among the C-terminal ligand-binding domain (2%–13% identity). However, pairwise structural superpositions show clear topological similarities among the C-terminal domains, notwithstanding differences in the lengths and relative orientations of α-helices.
The homodimeric arrangement of T1414 molecules found in the asymmetric unit shows that about 2197 Å2 (15.5%) of solvent-accessible surface area is buried on dimerization indicating that the dimer is functional. Across the two-fold axis of noncrystallographic symmetry, monomers superimpose with a RMSD of 0.55 Å for all Cα atoms. C-terminal domain helices H7 and H8 contribute to dimerization. The dimer association is mainly via the hydrophilic interactions, besides a few direct and water mediated hydrogen bonding contacts involving residues Ala114, Glu118, Arg121, Thr169, and Lys171 from helix H7 and H8 of both monomers.
Residues 28 to 47 of N-terminal domain encompass the DNA-binding region that includes a well-defined HTH motif (formed by helices H2 and H3), which is responsible for DNA binding in many other transcriptional regulators.26
The two helices pack together at an angle of ~ 85°, and are connected by a turn of five residues (Gly37–Ala38–Pro39–Lys40–Gly41). The HTH motif is stabilized by the strong hydrogen bond and van der Walls interactions with helix H1. The DNA-binding domain is structurally similar to those of TetR3
(pairwise RMSDs,1.1 Å). A superposition of the HTH motifs of T1414 (red), QacR (blue), and TetR (green) with accompanying bound DNA is shown in , revealing differences within the connecting turn region. Although the number of residues forming the HTH motif is same in all the three proteins, there are differences in the length of the so-called recognition helix (H3; four residues in T1414 and seven residues in both TetR and QacR) and in the number of residues comprising the turn motifs (five residues in T1414, four in TetR, and three in QacR). The side-chains of residues Leu43–Phe46 in the short recognition helix (H3) of T1414 adopt conformations essentially identical to those observed for corresponding residues in QacR and TetR. In both, TetR3
helix H3 makes extensive contacts with the major groove of bound DNA.
The observed sequence and structural similarity of the T1414 DNA-binding domain to those of QacR4
may provide insights into its DNA-binding properties. We anticipate that DNA recognition by T1414 is similar to that exhibited by TetR, which forms a dimeric interaction with DNA. Superposition of T1414 onto the QacR-DNA complex, suggests that the T1414 dimer uses the two HTH motifs to recognize two half sites within twofold symmetric DNA recognition elements found in the B. subtilis
The T1414–DNA model shows no obvious steric clashes between protein and DNA. The spacing between the two HTH motif of the dimer and the successive DNA major groove exactly complement each other. The modeled DNA engages both of the H3 recognition helices within the dimer. Calculated electrostatic potentials for the solvent accessible surfaces of TetR, QacR, and T1414 all show concentration positive features within the protein–DNA interfaces (both observed and predicted). Superficial T1414 residues in this area include those with positively charged side-chains, including Lys34 and Lys40. We believe, therefore, that T1414 binds DNA in a manner similar to that of QacR and TetR.
Comparison of ligand-binding pocket of TetR3
with T1414 suggests that the putative ligand-binding pocket is composed of residues from helices H5, H8, and H9 which form a narrow tunnel-like region. This tunnel, about 20 Å in length with variable diameter (4–6 Å), is predominantly lined by hydrophobic amino acids. Residues Ile56, Val59, Val66, and Leu69 from helix H5, Val123, Val127, Phe128, Trp131, and Phe135 from helix H8, and three leucines (152, 155, and 168) and four isoleucines (156, 160, 164, and 166) from helix H9 form the inner wall of the tunnel. The mouth of the tunnel has a positively charged patch, whereas the opposite end is negatively charged , both of which may act as charge-neutralizing regions for a bound, dipolar ligand. In our X-ray structure, the tunnel is occupied by a set of well-defined water molecules, which would be expelled on ligand binding. In QacR and TetR, similar tunnel regions support binding of six structurally diverse, cytotoxic drugs4
respectively. The identity of the T1414 binding ligand is not known at present.
Superposition of all six QacR–drug complexes with T1414 suggests that small molecules could bind to T1414 in a similar manner because the ligand-binding tunnels appear to be conserved. Specifically, the conserved aromatic/hydrophobic sidechains of Trp131, Phe87, Phe135, Ile84, Leu139, Met165, and Cys124 form the hydrophobic cluster in the putative drug-binding pocket of T1414. Superposition of the TetR–tetracycline complex11
with T1414 shows that tetracycline occupies an analogous hydrophobic region. These comparisons suggest that T1414 also bind small molecules in its putative ligand-binding pocket.