Neutrons probe structure of enzyme critical to development of next-generation HIV drugs

May 20, 2016

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 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.
-end-
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.

The paper is available at http://onlinelibrary.wiley.com/wol1/doi/10.1002/anie.201509989/full.

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.

UT-Battelle manages ORNL for the DOE's Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit http://science.energy.gov/.

DOE/Oak Ridge National Laboratory

Related HIV Articles from Brightsurf:

BEAT-HIV Delaney collaboratory issues recommendations measuring persistent HIV reservoirs
Spearheaded by Wistar scientists, top worldwide HIV researchers from the BEAT-HIV Martin Delaney Collaboratory to Cure HIV-1 Infection by Combination Immunotherapy (BEAT-HIV Collaboratory) compiled the first comprehensive set of recommendations on how to best measure the size of persistent HIV reservoirs during cure-directed clinical studies.

The Lancet HIV: Study suggests a second patient has been cured of HIV
A study of the second HIV patient to undergo successful stem cell transplantation from donors with a HIV-resistant gene, finds that there was no active viral infection in the patient's blood 30 months after they stopped anti-retroviral therapy, according to a case report published in The Lancet HIV journal and presented at CROI (Conference on Retroviruses and Opportunistic Infections).

Children with HIV score below HIV-negative peers in cognitive, motor function tests
Children who acquired HIV in utero or during birth or breastfeeding did not perform as well as their peers who do not have HIV on tests measuring cognitive ability, motor function and attention, according to a report published online today in Clinical Infectious Diseases.

Efforts to end the HIV epidemic must not ignore people already living with HIV
Efforts to prevent new HIV transmissions in the US must be accompanied by addressing HIV-associated comorbidities to improve the health of people already living with HIV, NIH experts assert in the third of a series of JAMA commentaries.

The Lancet HIV: Severe anti-LGBT legislations associated with lower testing and awareness of HIV in African countries
This first systematic review to investigate HIV testing, treatment and viral suppression in men who have sex with men in Africa finds that among the most recent studies (conducted after 2011) only half of men have been tested for HIV in the past 12 months.

The Lancet HIV: Tenfold increase in number of adolescents on HIV treatment in South Africa since 2010, but many still untreated
A new study of more than 700,000 one to 19-year olds being treated for HIV infection suggests a ten-fold increase in the number of adolescents aged 15 to 19 receiving HIV treatment in South Africa, according to results published in The Lancet HIV journal.

Starting HIV treatment in ERs may be key to ending HIV spread worldwide
In a follow-up study conducted in South Africa, Johns Hopkins Medicine researchers say they have evidence that hospital emergency departments (EDs) worldwide may be key strategic settings for curbing the spread of HIV infections in hard-to-reach populations if the EDs jump-start treatment and case management as well as diagnosis of the disease.

NIH HIV experts prioritize research to achieve sustained ART-free HIV remission
Achieving sustained remission of HIV without life-long antiretroviral therapy (ART) is a top HIV research priority, according to a new commentary in JAMA by experts at the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health.

The Lancet HIV: PrEP implementation is associated with a rapid decline in new HIV infections
Study from Australia is the first to evaluate a population-level roll-out of pre-exposure prophylaxis (PrEP) in men who have sex with men.

Researchers date 'hibernating' HIV strains, advancing BC's leadership in HIV cure research
Researchers have developed a novel way for dating 'hibernating' HIV strains, in an advancement for HIV cure research.

Read More: HIV News and HIV Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.