The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer

Abstract

The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix- turn-helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein-DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase alpha subunit.

Figure 1

Alignment of the LTTR and ModE transcription factor families. Sequence profiles of both families were aligned as described in Materials and Methods. The Figure shows 33 sequences selected from the profile–profile alignment. Sequences are designated by their SwissProt/trEMBL identifiers. An alignment of the E.coli OxyR and ModE proteins (OXYR_ECOLI, MODE_ECOLI) is identical to that computed by the SUPERFAMILY and MODBASE services. Shading was added to assist interpretation. Amino acids were divided into the following groups {FYVLIVMCA} (hydrophobic), {DE}, {KR}, {NQ} and {ST}. Residues belonging to the same group were considered similar and shading was added to indicate 100, 80 and 60% column similarity levels. The bottom line shows the secondary structure assignment according to the ModE structure (H, helix; E, strand). Secondary structure elements were labeled following the ModE structure.

Figure 2

Schematic ribbon diagrams of the structure of the full-length OxyR transcription factor dimer. (A) Homology model of the DBD. The HTH is marked in blue. Secondary structure elements are named according to Figure 1. (B) The full-length monomer of the OxyR reduced form. (C and D) The models of the oxidized (C) and reduced (D) forms of OxyR dimer bound to DNA (stereo images). Helix B in the regulatory domain is named according to Choi et al. (8). Helices B are located on the face of the cofactor binding domain dimer that is turned towards the dimer of the DBDs.

Figure 3

Hydrophobic core of the OxyR DBD (stereo view). Residues belonging to the hydrophobic core of the globular part of the DBD are colored red. The cluster of hydrophobic residues formed by helices α1 and α4 is colored green.

Figure 4

The electrostatic surface potential of the OxyR dimer. Electrostatic surface representation of the reduced form of the OxyR dimer with blue and red regions indicating positive and negative electrostatic regions, respectively. (A) The molecule shown in the same orientation as in Figure 2D. (B) The molecule is rotated by 90° around its long axis showing the face turned towards the DNA. Note the highly basic nature of the parts of the DBD which directly interact with the DNA. The negatively charged C-terminal part of recognition helix is marked by the square on both pictures.

Figure 5

Location of the residues which may act as contacts with αCTD. (A) Model of the DBD of the GcvA transcription factor. (B) Crystal structure of the DBD of the OmpR transcription factor. Note that in both structures the residues implicated in the contacts with αCTD are located in the turn of the HTH motif.