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

Alex Strom, Rachit Shah, Rafal Dolot, Melanie Rogers, Cher Ling Tong, David Wang, Youlin Xia, John D. Lipscomb, Carston R. Wagner

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state kcatand Kmfor turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate that KDalso 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.

Original languageEnglish (US)
Pages (from-to)2648-2661
Number of pages14
JournalBiochemistry
Volume61
Issue number23
DOIs
StatePublished - Dec 6 2022

Bibliographical note

Funding Information:
The authors acknowledge the financial support of this work from NIH Grants R01-AI146049 (to C.R.W.) and R35-GM118030 (to J.D.L.), as well as The American Foundation for Pharmaceutical Education Predoctoral Fellowship (to A.S.) and University of Minnesota Foundation (to C.R.W.).

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.

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