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Publication Abstract from PubMed

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 Zn(2+) 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 Zn(2+)-coordinated crystal structure are in fact part of the N-box alpha helix needed for efficient ATP binding. Such random-coil transformation of an N-box alpha helix turn and the closed-lid conformation are both artifacts arising from large millimolar Zn(2+) concentrations used in DosS CA crystallization buffers. In contrast, in the absence of Zn(2+), the short ATP-lid of DosS CA has significant conformational flexibility and can effectively bind AMP-PNP (K(d) = 53 +/- 13 muM), a non-hydrolyzable ATP analog. Furthermore, the nucleotide affinity remains unchanged when CA is conjugated to the DHp domain (K(d) = 51 +/- 6 muM). 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.

Understanding ATP Binding to DosS Catalytic Domain with a Short ATP-Lid.,Larson GW, Windsor PK, Smithwick E, Shi K, Aihara H, Rama Damodaran A, Bhagi-Damodaran A Biochemistry. 2023 Oct 31. doi: 10.1021/acs.biochem.3c00306. PMID:37905955^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.