Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a crucial enzyme for viral replication.
• Understanding the substrate binding mechanisms of HIV-1 RT is essential for developing effective antiviral therapies.
• HIV-1 RT contains 36 tryptophan residues, contributing to its intrinsic fluorescence properties.

Purpose of the Study:

• To investigate the binding characteristics of deoxynucleoside 5'-triphosphates (dNTPs) to the free HIV-1 RT enzyme.
• To determine the dissociation constants (Kd) for natural dNTPs and zidovudine triphosphate.
• To elucidate the nature of the dNTP binding site and the forces governing ligand association.

Main Methods:

• Utilized intrinsic tryptophan fluorescence quenching upon dNTP binding to measure binding affinities.
• Performed dual ligand titration experiments to assess competition and binding site characteristics.
• Calculated apparent dissociation constants (Kd) for various dNTPs and zidovudine triphosphate.

Main Results:

• Apparent Kd values for natural dNTPs ranged from 36.7 ± 1.8 μM (dTTP) to 47.3 ± 3.9 μM (dATP).
• Zidovudine triphosphate exhibited a Kd of 54.1 ± 1.3 μM, with no observed preference for purine or pyrimidine nucleotides.
• Identified a single dNTP binding site on the free enzyme, with hydrophobic interactions being the major driving force for ligand association (approx. 86% of binding energy).

Conclusions:

• The free HIV-1 RT enzyme possesses a single dNTP binding site that accommodates natural substrates and zidovudine triphosphate.
• Initial dNTP binding is primarily driven by hydrophobic forces, with electrostatic interactions contributing approximately 14% of the binding energy.
• Watson-Crick base pairing is not involved in the initial dNTP binding event, and Mg2+ is not essential for this process.