Neutrons probe structure of enzyme critical
to development of next-generation HIV
drugs
20 May 2016, by Jeremy Rumsey
A 3D structure of the HIV-1 protease in cartoon
representation with bound clinical drug darunavir (shown
as sticks). The catalytic site contains two closely
positioned aspartic acid residues. The insert depicts the
hydrogen transfer reaction in the catalytic site, captured
for the first time by neutron crystallography. Credit: Jill
Hemman and Andrey Kovalevsky, Oak Ridge National
Laboratory
A team led by the Department of Energy's Oak
Ridge National Laboratory used neutron analysis
to better understand a protein implicated in the
replication of HIV, the retrovirus that causes AIDS.
The enzyme, known as HIV-1 protease, is a key
drug target for HIV and AIDS therapies.
Researchers from ORNL, Georgia State University
and the Institut Laue-Langevin in France used
neutron crystallography
to uncover details of
interactions of hydrogen bonds at the enzyme's
active site, revealing a pH-induced proton 'hopping'
mechanism that guides its activity. The team
discussed the findings in a paper published in the
journal Angewandte Chemie.
Understanding the enzyme's structure and function
at the atomic level, including the location and
movement of
hydrogen atoms
, is vital for
understanding drug resistance and guiding rational
drug design.
HIV-1 protease is responsible for the maturation of
virus particles into infectious HIV virions, which
ultimately leads to the development of AIDS.
Without effective HIV-1 protease activity, HIV
virions remain non-infectious, so the disruption of
HIV-1 protease activity is a key target for
successful antiretroviral therapy (ART) drugs that
attack the virus itself.
The use of X-ray crystallography to study the
structures of HIV-1 protease and drug complexes
has led to the design of effective, commercially
available ART drugs, but x-rays cannot determine
the positions of mobile hydrogen atoms and
protons. Neutron crystallography, however, can
reveal these hydrogen-bonding interactions, which
play a key role in how effectively a drug binds to its
target.
The ORNL-ILL-Georgia State research team used
neutron crystallography to probe the structure of
HIV-1 protease in complex with the clinical inhibitor
Darunavir. The researchers combined neutron
diffraction data from the IMAGINE instrument at
ORNL's High Flux Isotope Reactor (HFIR), a DOE
Office of Science User Facility, and the LAD-III
instrument at ILL, to uncover details of the
hydrogen-bonding interactions in the active site and
reveal ways to enhance drug binding and reduce
drug resistance. The group also examined how the
enzyme's catalytic activity responds to changes in
pH (acidity) levels.
By determining structures at different pHs, the
group directly observed the positions of hydrogen
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atoms before and after a pH-induced two-proton
transfer between the drug and enzyme. The proton
transfer, triggered by electrostatic effects arising
from proton uptake by surface residues from
solution, resulted in the proton configuration that is
critical for the catalytic activity.
"These results highlight that neutrons represent a
superb probe to obtain structural details for proton
transfer reactions in biological systems," said ILL
instrument scientist Matthew Blakely.
"Darunavir's structure allows it to create more
hydrogen bonds with the protease active site than
most drugs of its type, while the backbone of HIV-1
protease maintains its spatial conformation in the
presence of mutations," said ORNL instrument
scientist Andrey Kovalevsky. "This means
Darunavir-protease interaction is less likely to be
disrupted by a mutation. Given these
characteristics, Darunavir is an excellent therapy
target to refine and therefore enhance HIV
treatment."
Direct observation of
proton transfer
in chemical
and biological systems is challenging;
macromolecular neutron crystallography has been
pivotal in providing key details regarding hydrogen
bonding that were required in order to answer long-
standing questions about the enzyme mechanism
of this important HIV drug target.
"Moreover, we observed changes in hydrogen
configurations induced by changes in protein
surface charges at long distances," said
Kovalevsky. "This phenomenon may occur in other
aspartic proteases, and perhaps in enzymes more
generally."
With the recent improvements that have been
made, the field of macromolecular neutron
crystallography is expanding, with studies
addressing a variety of important biological
processes from protein-folding to antibiotic
resistance and proton transport across biological
membranes.
Co-authors of the paper, titled "Long-Range
Electrostatics-Induced Two-Proton Transfer
Captured by Neutron Crystallography in an Enzyme
Catalytic Site," include lead author Oksana Gerlits,
Troy Wymore, Amit Das, Chen-Hsiang Shen, Jerry
M. Parks, Jeremy C. Smith, Kevin L. Weiss, David
A. Keen, Matthew Blakeley, John M. Louis, Paul
Langan, Irene T. Weber, and Andrey Kovalevsky.
Support for the preparation of deuterated HIV-1
protease was provided by the Center for Structural
Molecular Biology (CMSB). Both CMSB and the
research at HFIR were supported by DOE's Office
of Science. The IMAGINE instrument at HFIR is
funded by the National Science Foundation.
More information: Oksana Gerlits et al. Long-
Range Electrostatics-Induced Two-Proton Transfer
Captured by Neutron Crystallography in an Enzyme
Catalytic Site, Angewandte Chemie International
Edition (2016).
DOI: 10.1002/anie.201509989
Provided by Oak Ridge National Laboratory
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APA citation: Neutrons probe structure of enzyme critical to development of next-generation HIV drugs
(2016, May 20) retrieved 21 July 2018 from
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