Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/465575

Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis

Kumar, A.·Phulera, S.·Rizvi, A.·Sonawane, P.·Panwar, H. S.·Banerjee, S.·Sahu, A.·Mande, S. C.
View original source ↗Export metadata ↓

Abstract

The transcription factor Rv0081 of M. tuberculosis controls the hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis infection. We report here the crystal structure of Rv0081 at 3.3 [A] resolution revealing that it belongs to the well-known ArsR/SmtB family proteins. ArsR/SmtB family transcriptional repressors exert gene regulation by reversible metal binding. Hypoxia in general is sensed by bacterial transcriptional regulators via metals or Cys-mediated thiol switches. Oxygen sensing typically leads to transcriptional repressor changing its conformational state with altered DNA-binding property under different oxygen levels. Surprisingly Rv0081 neither has a metal binding domain nor does it possess Cys residues suggesting an alternate mechanism of gene regulation. Our structural analysis identified Ser 48, Ser 49, Ser 52 and Gln 53 as potential residues of Rv0081 involved in DNA binding. We probed DNA-binding of Rv0081 with electrophoretic mobility shift assay (EMSA) as well as surface plasmon resonance (SPR), where the Alanine mutants of these residues showed diminished DNA binding. Similarly, Aspartate mutants of these Ser residues was shown to fail to bind to DNA. Since, phosphorylation of various regulatory proteins is one of the important controlling mechanisms, we expected the role of Ser-phosphorylation of Rv0081 in hypoxic condition. Probing Rv0081 with anti-phosphoserine antibodies in M. tuberculosis cell lysate showed marked enhancement in the phosphorylation of Rv0081 protein under hypoxia. Overall, our structural and biochemical analysis provides the molecular basis for the regulation of Rv0081 in the latent phase of tuberculosis infection.\n\nIMPORTANCETuberculosis is one of the deadliest infectious diseases caused by the bacterium Mycobacterium tuberculosis. In about 90% of the infected people, M. tuberculosis exists in a dormant or a latent stage which can be reactivated in favorable conditions. Hypoxia (low oxygen pressure) is one of causes of dormancy. Understanding hypoxic gene regulation in M. tuberculosis is therefore an important step to understand latency. Rv0081 is a transcriptional regulator of genes expressed during hypoxia. In order to understand the mechanism by which Rv00081 regulates gene expression during hypoxia, we have solved the crystal structure of Rv0081 and identified amino acid residues which are critical in its transcriptional regulator activity. The crystal structure is suggestive of mechanism of gene regulation under hypoxia.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kumar, A., Phulera, S., Rizvi, A., Sonawane, P., Panwar, H. S., Banerjee, S., Sahu, A., Mande, S. C.. 2018-11-08. Structural basis of hypoxic gene regulation by the Rv0081 transcription factor of Mycobacterium tuberculosis. https://doi.org/10.1101/465575

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A Phenomenological Model of Synaptic Activity in the Blowfly Lamina

Estimates of the effective Poisson rate obtained from voltage recordings of Large Monopolar Cells (LMCs) in the blowfly lamina, measured in response to repeated light-contrast stimuli, are on the order of 5x105 events per second. These values imply that if vesicle exocytosis at photoreceptor-LMC synapse consisted of statistically independent quantal release events, the mean release rate would need to be at least this large to account for the observed signal and noise characteristics. Such a scenario is highly implausible, as it would correspond to an exocytosis rate of approximately 400 vesicles per second per active zone, or equivalently to the turnover of a membrane area comparable to that of all six photoreceptor axon terminals within a lamina cartridge more than twice per second. Relaxing the assumption of statistical independence, however, allows for substantially larger effective Poisson rates. In earlier work, we demonstrated that temporal regularization of release events- particularly through a reduction in low-frequency noise power spectral density- can increase the effective Poisson rate without altering the underlying mean release rate. Additionally, the contrast-to-current impulse response function of the extracellular space has been shown to be approximately equal in magnitude and opposite in sign to that of the LMCs, suggesting that both signals arise from the same chloride current. This observation provides a potential physiological mechanism for vesicle regularization, although it was previously described only in qualitative and simplified terms. In the present work, we develop a phenomenological model that incorporates these findings together with additional physiological parameters from literature to account for the regularization of vesicle release.

biophysics↗
bioRxiv2026

Massively parallel quantification and prediction of monomeric protein yield in Escherichia coli

Obtaining sufficient quantities of purified proteins in their intended molecular states is essential for biological research and protein engineering. Advances in computational protein design have expanded the functional capabilities of de novo proteins and accelerated candidate generation. However, reliable methods for ranking expected monomeric yield of candidate de novo designs, including molecular binders, fluorescent proteins and enzymes, have yet to be established. Standardized measurements of monomeric yield across diverse sequences remain scarce, limiting our understanding of its determinants and the development of accurate predictors for candidate selection. Here we developed a scalable pooled assay combining peptide barcodes, size-exclusion chromatography and mass spectrometry to quantify monomeric yield for over 39,000 natural protein domain variants, de novo proteins and nanobodies under uniform and standardized Escherichia coli production conditions. We used this dataset to (1) quantify the relationship between folding stability and monomeric yield, (2) benchmark inverse-folding methods, (3) evaluate the utility of structure-prediction confidence metrics in selecting high-yield design candidates, and (4) identify sequence and structural patterns in nanobodies associated with monomeric yield and monomer-to-dimer yield ratios. We further developed ESMC-MonoYield, a protein language model fine-tuned to predict monomeric yield from amino acid sequence alone, and prospectively demonstrated enrichment of high-yield candidates among BindCraft-designed binders. Our approach enables standardized, large-scale measurement of monomeric yield across diverse proteins, and establishes monomeric yield as a sequence-encoded property that can be learned, predicted and ultimately designed.

biophysics↗
bioRxiv2026

Functional Screening of Kv7.5 Channel Variants Associated with Neuronal Dysfunction

The KCNQ5 gene encodes Kv7.5 channels, which contribute to the regulation of neuronal excitability. Kv7.5 genetic variants associated with neurological dysfunction have been reported, but functional characterization of Kv7.5 variants is limited and causal links to pathogenicity are lacking. We have conducted the first automated patch clamp functional screen of a panel of heterozygous Kv7.5/Kv7.3 channel variants. We tested nine mutations located in diverse locations throughout the Kv7.5 channel and identified some variants with gain-of-function effects (L341I+/-, G347S+/-, P369R+/-, R497C+/-), some with loss-of-function effects (V145G+/-, W303C+/-), and others with no apparent effect (A131V+/-, R359H+/-, R491H+/-). Variants produced a gain of function by left-shifting the voltage-dependence of channel activation alone or in combination with increasing current density. Loss of function occurred as a result of reduced current density. Using these findings, we initiated the first z-score classification to guide understanding of Kv7.5 variant pathogenicity, which can be refined as more variants are functionally characterized and new variants are discovered. Lastly, we observed higher sensitivity to channel block by XE991 in L341I+/-, G347S+/-, P369R+/- gain-of-function variants indicating potential for targeted pharmacogenetic approaches to restore normal Kv7.5 channel function.

biophysics↗