1 research outputs found
Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides
Human histidine triad nucleotide-binding (hHINT) proteins
catalyze
nucleotide phosphoramidase and acyl-phosphatase reactions that are
essential for the activation of antiviral proTides, such as Sofosbuvir
and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit
disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial
activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic
surface residues (Q62, E100), which gate a conserved water channel
leading to the active site 13 Ã… away. hHINT2 crystal structures
identify analogous residues (R99, D137) and water channel. hHINT1
Q62 variants significantly alter the steady-state kcat and Km for turnover of
the fluorescent substrate (TpAd), while stopped-flow
kinetics indicate that KD also changes.
hHINT2, like hHINT1, exhibits a burst phase of adenylation, monitored
by fluorescent tryptamine release, prior to rate-limiting hydrolysis
and nucleotide release. hHINT2 exhibits a much smaller burst-phase
amplitude than hHINT1, which is further diminished in hHINT2 R99Q.
Kinetic simulations suggest that amplitude variations can be accounted
for by a variable fluorescent yield of the E·S complex from changes
in the environment of bound TpAd. Isothermal titration
calorimetry measurements of inhibitor binding show that these hHINT
variants also alter the thermodynamic binding profile. We propose
that these altered surface residues engender long-range dynamic changes
that affect the orientation of bound ligands, altering the thermodynamic
and kinetic characteristics of hHINT active site function. Thus, studies
of the cellular roles and proTide activation potential by hHINTs should
consider the importance of long-range interactions and possible protein
binding surfaces far from the active site