Determining the topology and ATP-Binding site of the central domain of TYRR by NMR

Full text: The protein TyrR belongs to the prokaryotic NtrC family of transcription factors that maintain metabolic homeostasis. TyrR, itself maintains aromatic ammo acid homeostasis by regulating the expression of proteins involved in the synthesis and transport of tyrosme, phenylalanine and tryptophan. TyrR differs from other members of this family in that it is not considered a 'two-component response regulator' and therefore does not require phosphorylation by another protein for activation. Furthermore, transcription activated by TyrR requires RNA polymerase with the ancillary subunit σ70 bound (Eσ 70 ) where other members require Eσ54. The proteins of the NtrC family including TyrR mostly consist of three domains an N-terminal domain that is unique to each member a C-terminal helix-turn-helix domain specific for its DNA site and a Central domain that shows distinct homology throughout the family. This latter domain has ATP-binding sites, an ATPase site, oligomenzation sites, and in the case of TyrR a tyrosine binding site. For other members of the NtrC family the Central domain has a site that interacts with Eσ54 while TyrR is thought to interact with Eσ70 through its N-terminal domain. Recently, we have had the goal of determining the structure of the Central domain of TyrR by NMR or Xray methods. Based on homology and limited proteolysis we have started with a 32 kDa construct. Preliminary analysis by NMR showed sample conditions were limiting and would require perdeuteration and TROSY methodology for assignment. Using a suite of 2H decoupled NMR experiments we have assigned the backbone resonances of the Central domain determined its secondary structure and topology. By following the effect of ATPγS titrations on 1H,15N resonances we have also determined the ATP binding site. These data strongly support the Central domain of the NtrC family belongs to a larger class of proteins referred to as the AAA+ superfamily of ATPases (1), which includes the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor (NSF)