M. tuberculosis Rv2623 is a nucleotide-binding protein 
We began a biochemical characterization of Rv2623 in order to gain insight into the relationship between the molecular structure/function of this USP and it's growth-regulatory properties.
M. tuberculosis Rv2623 was expressed in E. coli and purified to homogeneity for biochemical studies.
SDS-PAGE analysis of affinity-purified His6-Rv2623 revealed a single band that approximates the predicted molecular mass of approximately31.6 kDa, which was identified by immunoblotting as Rv2623 (Figure S3).
Gel filtration analysis of native His6-Rv2623 revealed that the purified protein exists primarily as a single species with an apparent molecular mass of 61+/-1 kDa; suggesting that Rv2623 is a dimer under native conditions (Figure S3), an observation that was later confirmed using nano electrospray ionization (nano ESI) mass spectrometry (data not shown).
The nucleotide-binding capacity of a subset of USPs was discovered following the observation that MJ0577, a single-domain USP from Methanococcus jannaschii, co-purifies and co-crystallizes with ATP [26].
On the basis of structures of ATP-binding and non-ATP-binding USPs, a G-2X-G-9X-G(S/T) motif was suggested to be essential for the binding of ATP [27].
The presence of this motif in each of the two tandem USP domains of Rv2623 [7] raised the possibility that this protein possesses ATP binding activity.
An HPLC-based examination of supernatants from boiled samples of His6-Rv2623 demonstrated that His6-Rv2623 co-purifies with both ATP and ADP (Figure 5).
Analysis of E. coli-expressed Rv2623 using nano ESI mass spectrometry also demonstrated that an ATP-saturated form of dimeric Rv2623 (composed of 2 bound ATP molecules per monomer) constitutes at least half of the purified sample (data not shown).
Measurement of the binding stoichiometry, which comprised HPLC-based quantification of adenine nucleotides from the boiled supernatant and spectral analysis of heat denatured Rv2623 following reconstitution in 6 M guanidine-HCl, yields 1.4+/-0.2 nucleotide equivalents/monomer with an overall content of 86+/-4% ATP (14+/-4% ADP).
Thus, Rv2623 binds endogenous adenine nucleotides in E. coli, and the association is sufficiently tight that nearly 75% of the nucleotide binding sites are occupied upon purification.
Indeed, nucleotide did not completely dissociate from the protein following an extensive, two-week dialysis with multiple changes against nucleotide-free buffer (approximately 0.3 nucleotide equivalents per monomer remain).
It is conceivable that the presence of ADP is the consequence of an Rv2623-associated ATP activity and this putative ATPase function is currently under investigation.
