HIV target: NMR revelations

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  • Published: Feb 17, 2014
  • Channels: NMR Knowledge Base
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Homing in on HIV

Structure of the MPER domain from HIV-1 gp41 envelope trimer in space filling format.  The binding sites of two HIV-1 broadly neutralizing antibodies are highlighted in blue for 2F5 and red for 4E10. Courtesy of Spicer et al

NMR spectroscopy has been used to home in on a vulnerable point in the HIV viral machinery, the envelope protein, gp41 membrane proximal external region. The research steals a march on other studies for which the protein is inaccessible, potentially leading the way to a vaccine or new targets for antiviral drugs against the disease.

The structure as determined by researchers at Duke University in Durham, North Carolina, could help focus the development of a vaccine against human immunodeficiency virus, the pathogen that causes AIDS. Such a vaccine has eluded medical research since AIDS was first positively identified in the early 1980s and its cause elucidated. The disease currently infects more than 33 million people worldwide and has killed more than 30 million. Details of the study were published online in an early edition of the journal Proceedings of the National Academy of Sciences (USA).

"One reason vaccine development is such a difficult problem is that HIV is exceptionally good at evading the immune system," explains team member Bruce Donald, a professor in Duke's computer science and biochemistry departments. "The virus has all these devious strategies to hide from the immune system." One of those strategies is that it undergoes drastic structural changes when it fuses to a host cell of the immune system. The envelope protein complex is a structure that protrudes from HIV's membrane and is responsible for infecting healthy host cells. Scientists have for several years targeted this complex for the development of a vaccine, and specifically its three copies of a protein called gp41 and closely associated partner protein gp120.

Trust in TROSY

The Duke team hopes that a particular region of gp41, called MPER, will be the virus's vulnerability, its Achilles' heel. "The attractiveness of this region is that, number one, it is relatively conserved," explains team member Leonard Spicer. In developing a successful vaccine against viruses as genetically variable as HIV, one must home in on a region that is conserved between variants, one that is very similar across all subtypes of the virus. The region in question has two particular sequences of amino acids that code for the binding of important broadly neutralizing antibodies. The HIV envelope region near the virus membrane is the point at which some of the most effective antibodies found in HIV patients bind and disable the virus.

When the virus fuses to a host cell, the HIV envelope protein transitions through at least three separate stages, the team explains. Its pre- and post-fusion states are stable and are well known to researchers, but it is the intermediate step - when the protein actually makes contact with the host cell - that has not been clarified, as it is so dynamic. The instability of this interaction has made it very difficult to visualize using traditional structure determination techniques, such as X-ray crystallography and even NMR spectroscopy.

The Duke team has used protein engineering and 600, 800, and 950 MHz nitrogen-15 TROSY (transverse relaxation optimized spectroscopy) HSQC (heteronuclear single quantum coherence) solution NMR spectroscopy and specially designed software to process limited data.

The trick to handling tricky

Duke's Patrick Reardon has spent many years engineering a protein that incorporates the HIV-1 MPER (membrane proximal external region), is associated with a membrane and behaves in a similar way to gp41 in the "tricky" intermediate step. This engineered protein is stable enough to divulge its secrets to the NMR spectrometer. The data captured the shape of the symmetric, MPER trimer in as state as close as possible to the native state.

The team validated the initial structure using an independent method of data analysis, which showed that alternative structures were inconsistent with the spectra. "The software took advantage of sparse data in a clever way that gave us confidence about the computed structure," Donald adds. It used advanced geometric algorithms to determine the structure of large, symmetric, or membrane-bound proteins - varieties that are very difficult to reconstruct from the NMR data.

But, the experiment does not end there. The protein must also demonstrate its ability to bind those broadly neutralizing antibodies. "One of the most important aspects of the project was ensuring that this construct interacted with the desirable antibodies, and indeed, it did so strongly," Reardon says.

"The next steps in this project are first to evaluate binding of the trimer to the calculated B-cell germline antibody and second to use the trimer structural information to design a potential vaccine candidate for HIV-1," Spicer told SpectroscopyNOW.

Related Links

Proc Natl Acad Sci, 2014, 111, 1391-1396: "Structure of an HIV-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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