Understanding ATP Binding to DosS Catalytic Domain with a Short ATP-Lid.

DosS is a heme-containing histidine kinase that triggers dormancy transformation inMycobacterium tuberculosis. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases reveals a short ATP-lid. This feature has been thought to block binding of ATP to DosS's CA domain in the absence of interactions with DosS's dimerization and histidine phospho-transfer (DHp) domain. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosS. We show that the closed-lid conformation observed in crystal structures of DosS CA is caused by the presence of Zn2+ in the ATP binding pocket that coordinates with Glu537 on the ATP-lid. Furthermore, circular dichroism studies and comparisons of DosS CA's crystal structure with its AlphaFold model and homologous DesK reveal that residues 503-507 that appear as a random coil in the Zn2+-coordinated crystal structure are in fact part of the N-box α helix needed for efficient ATP binding. Such random-coil transformation of an N-box α helix turn and the closed-lid conformation are both artifacts arising from large millimolar Zn2+ concentrations used in DosS CA crystallization buffers. In contrast, in the absence of Zn2+, the short ATP-lid of DosS CA has significant conformational flexibility and can effectively bind AMP-PNP (Kd = 53 ± 13 µM), a non-hydrolyzable ATP analog. Furthermore, the nucleotide affinity remains unchanged when CA is conjugated to the DHp domain (Kd = 51 ± 6 µM). In all, our findings reveal that the short ATP-lid of DosS CA does not hinder ATP binding and provide insights that extend to 2988 homologous bacterial proteins containing such ATP-lids.

Metalloprotein enabled redox signal transduction in microbes.

Microbes utilize numerous metal cofactor-containing proteins to recognize and respond to constantly fluctuating redox stresses in their environment. Gaining an understanding of how these metalloproteins sense redox events, and how they communicate such information downstream to DNA to modulate microbial metabolism, is a topic of great interest to both chemists and biologists. In this article, we review recently characterized examples of metalloprotein sensors, focusing on the coordination and oxidation state of the metals involved, how these metals are able to recognize redox stimuli, and how the signal is transmitted beyond the metal center. We discuss specific examples of iron, nickel, and manganese-based microbial sensors, and identify gaps in knowledge in the field of metalloprotein-based signal transduction pathways.