Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1

Abstract

Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.

Structure of theBacillus subtilisPhage SPO1-Encoded Type II DNA-binding Protein TF1 in Solution

“…We unambiguously identified several inter-and intramonomeric NOEs based on analysis of NOESY spectra of 2 H-labeled isotopic heterodimers (Jia et al, 1994). Those assignments were in agreement with the HU-based TF1 model.…”

“…The calculated secondary structure elements are essentially the same as reported based on the analysis of sequential NOE patterns (Jia et al, 1994) and the relative orientation of the secondary structure elements is very similar to the structure of the Bst. HU protein.…”

“…HU, and showed that the nine amino acid tail of TF1 adopts a kinked a-helical conformation (Reisman et al, 1993;Jia et al, 1994). We present here a refined solution structure of TF1 determined by multidimensional NMR techniques.…”

“…All distance constraints were extracted from a series of previously determined NOESY spectra (with mixing times of 75, 100, 150, and 200 ms) for each of eight selectively deuteriated TF1 variants (Jia et al, 1994). Effects of spin diffusion were monitored by comparing crosspeak intensities at each mixing time.…”

On the Connection Between Inherent DNA Flexure and Preferred Binding of Hydroxymethyluracil- containing DNA by the Type II DNA-binding Protein TF1

“…Two b-strands from each monomer extend into loops, or ''arms'', that can embrace a DNA helix resting on top of the b-strands (Tanaka et al, 1984;White et al, 1989). The high sequence homology between HU, IHF, and TF1 gives credence to the notion that these structural features apply to all three proteins (White et al, 1989) and this proves to be the case (Reisman et al, 1993;Jia et al, 1994;Nash, 1996). TF1 and IHF, both of which bind DNA sequence-selectively, differ from the HU proteins in having carboxy-terminal extensions: TF1 is a homodimer of 99 amino acid subunits, whereas IHF is a heterodimer of 98 and 94 amino acid subunits.…”