By David N. Leff

Would you believe the four-letter word that designates a human gene named ring stands for "really interesting new gene?"

This quixotic moniker was coined a decade ago by a British structural biologist, Paul Freemont, then at the Imperial Cancer Research Foundation in London.

"Freemont found," observed cancer molecular geneticist Frank J. Rauscher III, "that the RING domain was the common, conserved region in many different proteins. It contained cysteine amino acids at certain places in its sequence, suggesting it might chelate zinc.

"RING fingers," he added - "the small motif in the ring gene - are now found in 200 or more proteins, spread all over the animal and plant kingdoms. They're only about 60 amino acids long. Once you know what to look for in a small domain," he added, "you can simply search databases in silico - by computer - and find out how many there are, only you don't know their functions." (Domains are portions of proteins that exercise certain functions independently of, or cooperatively with, other domains of the protein.)

Rauscher, deputy director of the Wistar Institute's Cancer Center in Philadelphia, is co-senior author of a paper in the current EMBO Journal, dated Jan. 15, 2001. (This twice-monthly periodical is published by the European Molecular Biology Organization, in Germany.) His co-senior author is structural biologist Katherine Borden, an assistant professor of physiology and biophysics at the Mt. Sinai School of Medicine in New York.

Their article in EMBO bears the title: "Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains."

"The most important thing about these small domains," Rauscher told BioWorld Today, "is to identify their function and their structure. The first structure of a RING finger was accomplished by my co-author, Katherine Borden. She showed that this RING domain actually does chelate - bind to - the zinc ion, and as it folds around these two zinc molecules, it has a very common tertiary structure."

RING Finger Wraps Around Zinc, Not Gold

This arcane 3-dimensional atomic conformation has a critical bearing on human disease:

"It's now known for many RING finger proteins," Rauscher pointed out, "that in order to function, they must fold properly around the zinc molecules. In fact, the amino acids in the RING finger that contact zinc are absolutely required for that folding. As a consequence," he went on, "those amino acids are targets for mutation.

"The best example now," he recounted, "is the human BRCA-1 protein, which causes familial breast and ovarian cancer. It's a huge protein of 1,863 amino acids. Very often there's a single mutation in the tinier, 60-residue RING finger of BRCA-1 that converts one of its cysteine amino acids to something that won't contact zinc - leaving it completely unfolded. If a woman inherits that single amino acid change, her lifetime risk of getting breast cancer is over 80 percent. In nonfamilial mammary cancer, that risk is only 5 percent.

"The unique aspect of our present study," Rauscher said, "is that we took a similar domain, first discovered in the plant world. The plant homeodomain, PHD - like RING - has cysteine and histamine residues. PHD is also known as a leukemia-associated protein domain. It occurs in more than 400 different eukaryotic proteins throughout the plant and animal kingdoms. Many of these are believed to be involved in the regulation of gene expression. The PHD domain is a repressor, meaning that it turns genes off by silencing them. However PHD's structure was not known - until we solved it in this paper.

"The KAP-1 co-repressor," Rauscher went on, "is absolutely required for gene silencing - probably during embryonic development. We report in this paper, and another soon to come out, that if you make a point mutation in the PHD domain, you abolish that ability to silence genes. The dogma in the gene regulation field," he continued, "is that all of these proteins - like KAP-1 - are composed of many individual subdomains that fold independently. The PHD domain is one of them. In order to understand how to target that function pharmacologically, say with a small inhibitory molecule, you really need to have solved its tertiary structure."

The two collaborating laboratories, in Philadelphia and New York, were responsible for distinct aspects of the study. Rauscher and his co-authors at Wistar identified the PHD domain and its importance, and purified quantities of the PHD protein, using recombinant DNA technology.

Borden and her team at Mt. Sinai then used nuclear magnetic resonance (NMR) to analyze the structure of the molecule. She explained, "One region of the PHD domain latches on to the gene, much like planting one's feet on the ground, whereas using one's arms - the active region - does the work of turning a gene on or off."

"In a sense," Rauscher observed, "the regulatory molecules that determine which of the estimated 60,000 genes in any human cell are active or inactive at a given time, do as much as the genes themselves to define the character of that cell - whether it be skin, eye, liver or other type. Certainly," he went on, "precise gene regulation is critical for health, and flaws in these molecules have been linked to many serious medical conditions."

Goal: Mimic Or Muzzle PHD Domain Pathology

Pursuing this point, Rauscher added, "Naturally occurring point mutations or deletions of this domain contribute to a variety of human diseases, besides breast cancer. They include the ATRX mental retardation syndrome, childhood leukemias and autoimmune dysfunction. Clinically relevant missense mutations in the PHD domain of the ATRX protein," he pointed out, "result in truncated proteins where one or more of the PHD domains are deleted in patients with autoimmune polyglandular syndrome, head and neck squamous cell carcinomas and William's syndrome.

"Having in hand the molecular structure for this widely occurring gene switch," Rauscher suggested, "begins to help us explain why mutations in this molecule can lead to cancers and many other diseases. Our hope," he concluded, "is that we may now be able to design drugs to inhibit or mimic the function of the PHD molecule, when there are problems with it, or - ideally - even to rescue it to restore its proper function."