Mechanistic Studies of Bioorthogonal ATP Analogues for Assessment of Histidine Kinase Autophosphorylation

Phosphorylation is
an essential protein modification and is most
commonly associated with hydroxyl-containing amino acids via an adenosine
triphosphate (ATP) substrate. The last decades have brought greater
appreciation to the roles that phosphorylation of myriad amino acids
plays in biological signaling, metabolism, and gene transcription.
Histidine phosphorylation occurs in both eukaryotes and prokaryotes
but has been shown to dominate signaling networks in the latter due
to its role in microbial two-component systems. Methods to investigate
histidine phosphorylation have lagged behind those to study serine,
threonine, and tyrosine modifications due to its inherent instability
and the historical view that this protein modification was rare. An
important strategy to overcome the reactivity of phosphohistidine
is the development of substrate-based probes with altered chemical
properties that improve modification longevity but that do not suffer
from poor recognition or transfer by the protein. Here, we present
combined experimental and computational studies to better understand
the molecular requirements for efficient histidine phosphorylation
by comparison of the native kinase substrate, ATP, and alkylated ATP
derivatives. While recognition of the substrates by the histidine
kinases is an important parameter for the formation of phosphohistidine
derivatives, reaction sterics also affect the outcome. In addition,
we found that stability of the resulting phosphohistidine moieties
correlates with the stability of their hydrolysis products, specifically
with their free energy in solution. Interestingly, alkylation dramatically
affects the stability of the phosphohistidine derivatives at very
acidic pH values. These results provide critical mechanistic insights
into histidine phosphorylation and will facilitate the design of future
probes to study enzymatic histidine phosphorylation.