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

Publication Type:

Journal Article


Biochemistry, Volume 62, Issue 22, p.3283-3292 (2023)


Adenosine Triphosphate, Bacterial Proteins, Catalytic Domain, Histidine, Histidine Kinase, Protein Conformation


<p>DosS is a heme-containing histidine kinase that triggers dormancy transformation in. 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&#39;s CA domain in the absence of interactions with DosS&#39;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 in the ATP binding pocket that coordinates with Glu537 on the ATP-lid. Furthermore, circular dichroism studies and comparisons of DosS CA&#39;s crystal structure with its AlphaFold model and homologous DesK reveal that residues 503-507 that appear as a random coil in the Zn-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 Zn concentrations used in DosS CA crystallization buffers. In contrast, in the absence of Zn, the short ATP-lid of DosS CA has significant conformational flexibility and can effectively bind AMP-PNP ( = 53 &plusmn; 13 μM), a non-hydrolyzable ATP analog. Furthermore, the nucleotide affinity remains unchanged when CA is conjugated to the DHp domain ( = 51 &plusmn; 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.</p>