The Catalytic Mechanism
of HIV‑1 Integrase
for DNA 3′-End Processing Established by QM/MM Calculations
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Abstract
The development of HIV-1 integrase (INT) inhibitors has
been hampered
by incomplete structural and mechanistic information. Despite the
efforts made to overcome these limitations, only one compound has
been approved for clinical use so far. In this work, we have used
all experimental information available for INT and similar enzymes,
to build a model of the holo-integrase:DNA complex that includes an
entire central core domain, a ssDNA GCAGT substrate, and two magnesium
ions. Subsequently, we used a large array of computational techniques,
which included molecular dynamics, thermodynamic integration, and
high-level quantum mechanics/molecular mechanics (QM/MM) calculations
to study the possible pathways for the mechanism of 3′ end
processing catalyzed by INT. We found that the only viable mechanism
to hydrolyze the DNA substrate is a nucleophilic attack of an active
site water molecule to the phosphorus atom of the scissile phosphoester
bond, with the attacking water being simultaneously deprotonated by
an Mg<sup>2+</sup>-bound hydroxide ion. The unstable leaving oxoanion
is protonated by an Mg<sup>2+</sup>-bound water molecule within the
same elementary reaction step. This reaction has an activation free
energy of 15.4 kcal/mol, well within the limits imposed by the experimental
turnover. This work significantly improves the fundamental knowledge
on the integrase chemistry. It can also contribute to the discovery
of leads against HIV-1 infection as it provides, for the first time,
accurate transition states structures that can be successfully used
as templates for high-throughput screening of new INT inhibitors