Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in Mycobacterium tuberculosis.

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1.0 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1 s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1 s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC). This method can be performed in anaerobic glove bag settings, requires readily available HPLC instrumentation for subsequent detection, and is adaptable to meet the needs of a variety of substrate affinity measurements.

Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in Mycobacterium tuberculosis.

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.

Molecular engineering of biocatalysts has revolutionized complex synthetic chemistry and sustainable catalysis. Here, we show that it is also possible to use engineered biocatalysts to reprogram signal transduction in human cells. More specifically, we manipulate cellular hypoxia (low O2) signaling by engineering the gas-delivery tunnel of prolyl hydroxylase 2 (PHD2), an iron-dependent enzymatic O2 sensor. Using computational modeling and rational protein design techniques, we resolve PHD2's gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. Systematic modification of these residues open the constriction topology of PHD2's gas tunnel with the most effectively designed mutant displaying 11-fold enhanced hydroxylation efficiency. Furthermore, transfection of plasmids that express these engineered PHD2 mutants in HEK-293T cells reveal significant reduction in the levels of hypoxia inducible factor (HIF-1α) even under hypoxic conditions. Our studies reveal that activated PHD2 mutants can reprogram downstream HIF pathways in cells to simulate physiological O2-like conditions despite extreme hypoxia and underscores the potential of engineered biocatalysts in controlling cellular function.

Understanding ATP binding to DosS catalytic domain with a short ATP-lid.

DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS's CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP (Kd = 53 ± 13 µM). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.

Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases.

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket. Detailed computational investigations indicate that a negatively charged glutamate hydrogen-bonded to iron's equatorial-aqua ligand acts as an electrostatic lock preventing both lysine and anion binding in the absence of the other. Using a combination of UV-Vis spectroscopy, binding affinity studies, stopped-flow kinetics investigations, and biochemical assays, we explore the implication of such active site assembly towards chlorination, bromination, and azidation reactivities. Overall, our work highlights previously unknown features regarding how anion-substrate pair binding govern reactivity of iron halogenases that are crucial for engineering next-generation C-H functionalization biocatalysts.

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins.

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.