Last Month's Most Accessed Feature: Final piece of protein puzzle: NMR tops and tails HIV

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  • Published: Dec 1, 2017
  • Categories: NMR Knowledge Base
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Piecing together HIV

Cytoplasmic tail of gp41 The structures of three membrane-associated cytoplasmic tails of gp41 are shown in red and orange, from this illustration of the trimeric spike structure. The rest of this HIV-1 envelope protein, which is docked on a gray membrane, are the structures of gp120 and gp41, shown in green, and the transmembrane segment, shown in blue. Credit: Jamil Saad

Nuclear magnetic resonance (NMR) spectroscopy has been used to put in place the final piece of the HIV-1 protein structure, the cytoplasmic tail of gp41 protein. The research by a team at the University of Alabama at Birmingham led by Jamil Saad will help scientists better understand how the virus infects human cells and how progeny viruses are assembled and released from infected cells leading to acquired immune deficiency syndrome (AIDS).

The cytoplasmic tail of gp41 is a critical piece of the AIDS virus. It appears to help incorporate the envelope spike structures into the surface of viral particles during viral assembly. "If we are able to inhibit incorporation of the envelope protein, we inhibit viral replication,” Saad explains. “This would disarm the virus and prevent disease. The cytoplasmic tail is a critical component of infectivity.” Thus there has been considerable effort expended in studying the structure of the cytoplasmic tail of gp41 but it has proved rather difficult to solve. "It was the most challenging project I have encountered in the past 20 years," Saad says. "Many times I was about to quit, but I have a very persistent lab group."

Structurally stymied

There have been many research group stymied by the complexity of this piece of protein. One problem that arises is that expressing the cytoplasmic tail of gp41 in the laboratory from engineered Escherichia coli cells, for instance is difficult. Instead of intact product, Saad's team found that one bacterial enzyme was cutting the protein during protein expression. “We have never seen this for any other protein,” Saad explains. The team found that they had to limit protein expression to just two hours. Even then, 70 percent of the cytoplasmic tail was cut and only 30 percent was left intact. As such, it took them more than two years and efforts with 100 different reaction conditions to obtain adequate product of sufficient integrity to be used in an NMR experiment. Of course, Saad adds that these days they can express the protein well in just two days.

Indeed, the enzymatic cut turned out to be a useful phenomenon. The team found that one fragment was soluble in water, and so could readily be examined by NMR spectroscopy. The other fragment was lipophilic and so had to be dispersed using micelles to mimic a membrane-like environment. It took a lot of trial and error to find the right growing conditions to generate this fragment. Nevertheless, NMR spectroscopy was also able to provide a structure for the insoluble fragment of the protein while incorporated into the micelles. Indeed, the team put molecules of the intact, full-length cytoplasmic tail of gp41 into the micelle solution, they found that the structures of each of the pieces were maintained in the intact structure.

A tail to tell

They demonstrated that the N-terminal, just 45 amino acid residues, lacked a regular secondary structure and was not associated with the membrane, which wraps around an HIV-1 viral particle. The C-terminal, a chunkier 105 amino acid residues, was shown to be tightly associated with the membrane and had three alpha-helixes with portions that were hydrophobic and portions that were hydrophilic. The study also gave the group a picture of the preferred topology of the cytoplasmic tail when bound to the membrane.

The researchers suggest that their structure could open a new area of research into HIV-1. For instance, it points to how the cytoplasmic tail stabilizes the envelope protein structure, how it affects membrane mobility of the envelope protein structure, and how it helps coordinate membrane-binding and the association of 2,000 HIV-1 Gag polyproteins underneath the membrane surface. The Gag polyproteins are later cut inside the maturing virus to form multiple smaller matrix, capsid and nucleocapsid proteins that produce the condensed viral core.

“The cytoplasmic tail of gp41 has been of interest for a long time, and nobody understands how it functions in infection or how it helps incorporate the envelope protein into the membrane,” Saad said. The new knowledge will lead to new comparative studies of ten different retroviruses that have similar tails to learn why some tails are shorter and some tails are longer.

Related Links

Structure 2017, online: "Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein"

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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