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Seminar Highlights a Promising HIV Inhibitor

By Robin Arnette
January 2009

Angela M. Gronenborn, Ph.D.
Gronenborn, who was elected to the prestigious National Academy of Sciences in 2007, is the Rosalind Franklin professor and chair of the Department of Structural Biology at the University of Pittsburgh Medical Center (UPMC). (Photo courtesy of Angela Gronenborn and UPMC)

Structural biologists use techniques such as X-ray crystallography, mass spectrometry and nuclear magnetic resonance (NMR) to determine the structure and function of molecules. One such specialist visited NIEHS on May 7 to talk about her work on cyanovirin-N (CV-N), an unusual protein that inhibits the human immunodeficiency virus-1 (HIV-1) - the causative agent of AIDS. Angela M. Gronenborn, Ph.D., presented "The CVNH Family of Lectins: Structure, Folding and Sugar Binding" as part of the NIEHS Laboratory of Structural Biology (LSB) Seminar Series. Kelly Mercier, Ph.D., a fellow in the LSB Nuclear Magnetic Resonance (NMR) Group hosted the lecture.

Over the course of her career, Gronenborn ( Exit NIEHS has studied a large number of proteins, but she chose to focus on one serendipitous molecule for her talk. "Mike Boyd at NCI was screening extract from the cyanobacterium Nostoc ellipsosporum for anti-HIV activity and found something interesting, a protein now called cyanovirin-N (CV-N)," she said. "I attended an NMR meeting [afterwards], and a colleague asked me to look at the spectrum of this protein. At the time, I didn't know what it was, but I was determined to find out."

Other scientists had concluded that CV-N was stable under stringent conditions. During the purification process, the researchers treated it with alcohol and 0.1 percent sodium dodecyl sulfate (SDS) and boiled it, but it still had activity. The team eventually ascertained that it was 11 kDa with two disulfide bonds, but the most fascinating finding was that it wasn't related to any sequences in the protein sequence database.

Gronenborn furthered the work by using NMR to determine that the protein was a monomer in solution, made up mostly of β-sheets in a completely new fold - no similar structure had been deposited in the Protein Structure Database (PSdb). Surprisingly, X-ray crystallography showed a domain-swapped dimer in the crystal state. Gronenborn explained that domain swapping produces an intertwined dimer when a domain of monomer A is exchanged with an identical domain of monomer B. To her amazement, additional experiments revealed that CV-N was also able to adopt three different confirmations depending on the environment. "When you pull up the coordinates of a protein from the PSdb," Gronenborn warned, "just know that the structure you see exists under the conditions at which the data were collected; there may be alternative confirmations."

Because of its anti-viral activity, CV-N is an important player in the fight against AIDS. Virologists had discovered that HIV has a protein on its surface called envelope glycoprotein (gp120) that is highly glycosylated, which means it contains many sugar molecules. If researchers add CV-N to a mix of human cells and HIV, the CV-N binds to the sugars on the gp120 and prevents the virus from infecting the cells. Gronenborn used NMR to map the binding site of the sugar on CV-N and also found that the 3' and 4' hydroxyl groups on terminal mannose units on the sugar are crucial for the interaction.

The research team spent several years doing this work, and during that time, sequences of CV-N homologues from other organisms were deposited into the gene bank, including ferns and two species of fungi - the Tuscany White Truffle (Tuber borchii) and the red bread mold (Neurospora crassa). Gronenborn examined the structure of these related versions of CV-N and found that they have slight variations in structure, but a similar architecture overall.

Gronenborn concluded her seminar by saying another scientist discovered that the N. crassa CV-N was related to a Clock gene, which is responsible for the 24-hour cycle of circadian rhythms in living organisms. She said, "At least in N. crassa, we know the protein has something to do with the biorhythms of an organism, but in the others we haven't a clue."

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