Structural Modeling of NTPDase-Substrate Complexes Preserving Catalytic Experimental Features.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzymes regulate purinergic and pyrimidinergic signaling by hydrolyzing nucleoside phosphates.
• E-NTPDases hold significant therapeutic potential, but limited structural insights into their substrate complexes hinder enzyme optimization.
• Existing molecular docking methods often fail to accurately represent experimentally observed substrate conformations.

Purpose of the Study:

• To develop a computational strategy for modeling E-NTPDase-substrate complexes that preserves experimentally validated substrate features.
• To leverage conserved active site features across the E-NTPDase family for accurate modeling.
• To generate reliable structural models of human E-NTPDases (HsNTPDases) complexed with various nucleotide substrates.

Main Methods:

• Developed a computational strategy integrating conserved active site characteristics with experimentally observed substrate conformations.
• Identified a canonical linear-like substrate conformation common across E-NTPDase structures, including the phosphate tail and nucleobase.
• Applied the method to model Homo sapiens NTPDases (HsNTPDase1-8) with ATP, ADP, GTP, GDP, UTP, and UDP.

Main Results:

• The computational strategy successfully modeled HsNTPDases complexed with multiple nucleotide substrates.
• Models accurately positioned essential metal ion cofactors and catalytic water molecules within the active site.
• The identified canonical substrate conformation provides a conserved feature for accurate enzyme-substrate interaction studies.

Conclusions:

• The developed computational approach offers a reliable framework for studying E-NTPDase-substrate interactions.
• These accurate models facilitate rational enzyme engineering for therapeutic applications.
• The findings pave the way for advancing the therapeutic exploration of E-NTPDases.