NMR studies of a hypothetic protein from Xanthomonas axonopodis pv. citri suggests that it could be a transcription regulator involved in quorum sensing
Roberto K. Salinas; Lucicleide Ribeiro and Chuck S. Farah
Biochemistry Department, IQ - USP, Sao Paulo, SP
The open reading frame 1883 of the phytopathogen Xanthomonas citri's main chromosome is adjacent to the rpf locus that controls quorum sensing in Xanthomonas.
It codes for an 82-residue protein of unknown function, XAC1883,
containing a SpoVT/AbrB domain (Pfam family 04014) in its amino
terminus (residues 3-48). The best characterized member of this family,
AbrB from Bacillus subtilis, is a DNA binding protein known to
control the transcription of genes involved in the control of
stationary phase functions and sporulation. Therefore, in Xanthomonas,
XAC1883 could be a transcription regulator that acts on promoters of
genes involved in quorum sensing pathways. In order to gain insight
into XAC1883 function, we are studying its solution structure by high
resolution NMR. Backbone resonance assignments are being carried out
using standard triple resonance NMR experiments combined with the
analysis of 15N edited TOCSY and NOESY spectra acquired with 15N labeled and 13C/15N doubly labeled samples. As much as 88 % of the backbone resonances were assigned. Residues Glu7, Gln11 and Lys54-Val58 are not yet assigned as their HN cross peaks are missing in the 15N HSQC spectrum possibly due to exchange broadening. The resonances of Ala6, Arg8 and Gly9 are only observable at higher temperature (35 0C),
suggesting that part of the N-terminal segment of the protein undergoes
conformational exchange at the NMR time scale. Nevertheless, secondary
structure predictions based on chemical shifts indicate the presence of
two short beta-strands at the N-terminus (residues 2 - 6 and 11 - 13), followed by an alpha-helix (residues 15 to 21), two more other beta-strands
(residues 23-29 and residues 36-42) and a short helical segment between
residues 46-49, while the C-terminal portion, residues 50-82 is
predicted to be unstructured. These predictions were confirmed by the
analysis of short range HN-HN and HN-Ha NOEs which are indicative of beta-sheet or alpha-helix conformations. Experiments to test whether XAC1883 binds to specific promoter DNA sequences are being initiated.
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