ppat-1000460-g005: 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).

Mentions: 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.

Drumm JE, Mi K, Bilder P, Sun M, Lim J, Bielefeldt-Ohmann H, Basaraba R, So M, Zhu G, Tufariello JM, Izzo AA, Orme IM, Almo SC, Leyh TS, Chan J - PLoS Pathog. (2009)

Bottom Line: Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity.Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity.We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

Affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.

Abstract: Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.