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 ∼31.6 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.
10.1371/journal.ppat.1000460.g005 Figure 5  M. tuberculosis Rv2623 is a nucleotide-binding USP.
High Performance Liquid Chromatography (HPLC) analysis of endogenously bound nucleotides from purified His6-Rv2623. Nucleotides species were identified based on their specific retention times on the Mono Q HR 5/5 column, represented by peaks in absorbance at 260 nm, which correspond to that of nucleotide standards (not shown). Bound nucleotides were extracted by boiling, and separated and quantified from a standard curve that relates absorbance peak area to the known amount of ATP injected onto the column (inset).

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