Near-atomic-level Details of Cancer Proteins with the Help of Molecular Cages Revealed

Sandwiching the wobble protein between two other layers allowed scientists to get the most detailed picture of a protein that is key to the spread of acute myeloid leukemia. Acute myeloid leukemia (AML) afflicts more than 20,000 Americans each year, killing more than 11,000 of them, according to the American Cancer Institute. And many people who receive intensive chemotherapy or stem cell transplants can experience side effects such as infection, hair loss and vomiting, as well as long-term complications.

Kathleen Sakamoto, a professor at Stanford University School of Medicine who has been working on developing drugs to treat acute myeloid leukemia and other blood disorders, is trying to address the situation. However, her team's search for new treatments for acute myeloid leukemia was hampered by a subtle gap between two techniques used to understand protein structure and function—X-ray crystallography on the one hand, and cryo-electron microscopy (cryo-EM) on the other.

Researchers from Stanford University School of Medicine and Engineering and the SLAC National Accelerator Laboratory at the Department of Energy have discovered a way to close the gap, using molecular cages to stabilize certain medium-sized proteins so that they can be imaged with cryo-electron microscopy for the first time, which can reveal almost atomic-level details.

The problem, says SLAC and Stanford professor Soichi Wakatsuki, is KIX, part of the CREB-binding protein (CBP) that AML cancer cells use to transcribe genes important for growth and survival. If researchers better understand its structure, they can design drugs that inhibit KIX, preventing cancer cells from replicating. But efforts to study the protein using X-ray crystallography have not been successful. The molecule's relatively large size by crystallographic standards makes it more difficult to crystallize, and even if it does, the peculiarities of the process make it harder to analyze the part of KIX that drug designers want to target.

At the same time, the KIX itself is a bit too small to be effectively studied with cryo-EM. Wakatsuki explained that in order to get a good image of a protein with cryo-EM, one has to be able to find many copies of the protein in the electron microscope image, and then figure out their orientation, whether they're curved in one way or another, etc. Only by finding and arranging many images of a protein can cryo-EM methods produce high-resolution structures. The relatively small size of KIX, by cryo-EM standards, makes it a challenge. Another option, Nmr, has been used to determine the structure of KIX when bound to other naturally-occurring molecules, but the method requires extensive preparation and analysis, making it less suitable for rapidly determining the structure of molecules, and therefore less suitable for studying the effects of potential KIX-inhibiting drugs.

The researchers came up with this solution over lunch when they were working on a separate project. They sandwiched batches of KIX proteins between a central globular molecule and an outer molecular cage. Because this "double shell" is much larger than a single KIX molecule, it will be easier to spot and localize in cryo-EM images, which will make it easier to obtain high-resolution images of the KIX molecule itself.

In addition to seeing the structure of KIX, Wakatsuki said, his lab and Chiu's lab, in collaboration with Sakamoto and Stanford computer science professor Ron Dror, were able to add other molecules to the mix to see if they might bind to KIX and potentially inhibit its function. The team reports that they have been able to increase the strength of this binding by about 200 times, which could help scientists develop drugs that are effective at lower doses.

The team's results also suggest that the method could prove useful for other proteins in between that are difficult to study with cryo-EM or X-ray crystallography, perhaps including some viral proteins. "We are moving forward to expand the applicability of the method," Wakatsuki said.