Uncovering Elusive Proteins

Stephen Blacklow describes the shape of CD81, the first protein of its kind to have its molecular structure completely revealed. Video: Stephanie Dutchen

Half-buried in the oily membranes of our cells lurk elusive proteins called tetraspanins.

Although tetraspanins perform a variety of essential jobs and are being linked to an increasing number of diseases, researchers don’t understand how they work—or break down—because they haven’t been able to figure out what the proteins look like.

“Tetraspanins are incredibly important in biology, and we know bloody little about them,” said Stephen Blacklow, chair of the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School.

“If you really want to understand how anything in biology works, you have to see what it looks like at the molecular level,” added Andrew Kruse, HMS assistant professor of biological chemistry and molecular pharmacology.

Now, Blacklow, Kruse and colleagues have pierced the veil surrounding tetraspanins by determining the first complete structure of one: tspan28, also known as CD81, a protein that helps B-cells mature for a robust immune system.

The researchers describe CD81’s surprising shape, not dissimilar to an ice cream cone that can hold a cholesterol molecule, in the journal Cell on Oct. 27.

The finding reveals that the part of CD81 previously hidden within the cell membrane is more important than many scientists assumed. The study also suggests that CD81, whose malfunction can lead to immune deficiencies and whose presence is necessary for hepatitis C infection, might make a useful drug target and provides clues for how to manipulate the protein.

“CD81 was predicted to be kind of useless. Instead, we found this binding pocket that no one expected,” said the study’s first author, Brandon Zimmerman, a former research fellow in the Blacklow lab who now works at Vertex Pharmaceuticals in Boston. “Knowing the structure opens the door to, in a fantasy, maybe one day being able to target this and other tetraspanins for clinical applications.”

The clinical potential is uncertain but broad, the researchers say, as studies in humans and mice have linked abnormal tetraspanin function to cancer, diabetes, bleeding disorders, kidney and skin diseases, eyesight and hearing loss, sterility and more.

Oil and water

Like carrots rooted in soil with only their greens sprouting above ground, tetraspanins have an inner, transmembrane portion and an outer, extramembrane portion.

In the 25 years following the discovery of tetraspanins, scientists caught a glimpse of the isolated outer segment. The oily transmembrane segment, however, and its relationship to the outer segment, remained shrouded in obscurity because it doesn’t cooperate with common structure-deciphering tools, which are water-based.

Some researchers tried to pry into the black box and failed. Others shrugged, figuring the transmembrane portion wasn’t important but merely served as an anchor for the outer segment.

“Structural studies of membrane proteins were very daunting up until about ten years ago,” said Blacklow, senior author of the paper and the Gustavus Adolphus Pfeiffer Professor of Biological Chemistry and Molecular Pharmacology at HMS.

The HMS-led team bet on co-author Kruse’s expertise in a technique called lipidic cubic phase crystallography. The method allows researchers to crystallize and capture the structures of membrane proteins while they’re embedded in an oily matrix that mimics their natural environment.

The wager paid off, and the researchers got their first full look at a tetraspanin—including some unique and unexpected features.

“Brandon’s perseverance and biochemical skills and Andrew’s determination and deep knowledge of membrane protein structural biology made it possible,” said Blacklow.

“Some luck too,” Zimmerman added.

Hidden pocket

Tetraspanins get their name from crossing the cell membrane four times. The team was surprised to find that CD81’s four main coils weren’t all bundled together as expected but instead divided into two pairs that come together on one end to form a cone.

“It’s different from all prior predictions,” said Blacklow.

The second surprise came with the unearthing of CD81’s inner cavity and the discovery that it can hold a cholesterol molecule. That got the team especially excited, because it revealed that researchers can now try to manipulate CD81 using cholesterol or other small molecules.

“No one thought the transmembrane portion was important,” said Zimmerman. “Clearly that isn’t the case.”

Computer simulations run by collaborators at Stanford University revealed that when bound to cholesterol, CD81 sits around with its extramembrane cap closed, but when unbound, its cap occasionally flips open.

Additional experiments led the team to propose that abundant cholesterol holds CD81 closed. That makes it harder for CD81 to interact with other proteins, including the tetraspanin CD19, which is needed for B-cells to properly develop and function.

Conversely, in a cholesterol-poor environment, such as the endoplasmic reticulum or ER, where CD19 is produced, the researchers suspect that CD81 can open up and ferry CD19 to the cell surface, where it then becomes part of the B-cell receptor—a core element of the immune system’s response to pathogens.

The findings suggest that “you could either give a drug that mimics cholesterol and holds CD81 closed or give a drug that interferes with cholesterol binding and causes it to be open,” said Zimmerman. “Both avenues could have therapeutic importance.”

One area of interest, the researchers say, is immune-based cancer treatments called CAR T-cell therapies that target B-cell cancers by homing in on CD19.

“Like they say in ‘Forrest Gump,’ life—or structural biology—is like a box of chocolates,” said Blacklow. “You never quite know what you’re going to get.”

“But it’s almost always interesting,” said Kruse.

Illuminating dark matter

In addition to helping researchers understand and potentially target CD81, the discovery may illuminate other tetraspanins and their disease connections because they likely share CD81’s structure, the researchers say.

For example, the work could assist researchers who are taking aim at the tetraspanin CD37 in clinical trials to treat chronic lymphocytic leukemia and non-Hodgkin lymphoma.

Even if clinical applications don’t pan out, though, the work excites Blacklow because it represents one of the last remaining opportunities to illuminate “dark matter” in transmembrane protein biology, he said.

“A lot of times, we elucidate some new detail or facet of a particular molecule or process,” he said. “There are fewer and fewer completely new things that we get to see.”