A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Abstract

Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L-and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L-and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.

Subunit Vaccines Consisting of Antigens from Dormant and Replicating Bacteria Show Promising Therapeutic Effect against Mycobacterium Bovis BCG Latent Infection

“…These three proteins were shown to be upregulated in hypoxic conditions of in vitro M. tuberculosis latency models [33,34]. Rv1738 was found to inhibit the ribosomal protein synthesis in order to induce M. tuberculosis non-replicating persistence [35]. The expression of Rv2626c increased at the terminal stages of M. tuberculosis infection [36,37].…”

Ribosome profiling inMycobacterium tuberculosisreveals robust leaderless translation

“…Analysis of the differential expression of ribosome-associated factors during starvation highlighted the significant up-regulation of two genes, Rv1738 and RafS (DESeq2, (Love et al, 2014)). The function of the product of gene Rv1738 has not been biochemically determined, but structural studies suggest it may function as a ribosome hibernation promoting factor (HPF) as it bears significant structural homology to the HPF proteins from E. coli and Vibrio cholerae, including docking of the Rv1738 protein dimer into the groove where HPFs bind the 70S ribosome (Bunker et al, 2015). The up-regulation of Rv1738 in starvation is consistent with its potential role as an HPF and with a role in tolerance of early nutrient starvation.…”

“…The up-regulation of Rv1738 in starvation is consistent with its potential role as an HPF and with a role in tolerance of early nutrient starvation. Rv2632c bears some homology to Rv1738, but lacks the ring of positively charged residues likely to be important for ribosomal RNA binding (Bunker et al, 2015). Rv2632c was also up-regulated in starvation (log2 fold-change = 1.59) but the up-regulation fell below the threshold of significance (log2 fold-change = 2).…”

Racemic crystal structures of peptide toxins, GsMTx4 prepared by protein total synthesis

“…With well‐folded GsMTx4 protein in hand, we began to perform the racemic crystallization protocol to obtain the X‐ray structure. Racemic crystallization is a powerful technique that has been widely used for the crystallization of toxin proteins . L‐ and D‐GsMTx4 were then mixed to a final concentration of 1.5 mg/mL and subjected to crystal screening in 18°C crystal chamber using a Hampton Crystallization Kit.…”