Mechanism of action of melanoma protein B-Raf

It starts off small, just a skin blemish. The most common moles stay just that way -- harmless clusters of skin cells called melanocytes, which give us pigment. In rare cases, what begins as a mole can turn into melanoma, the most serious type of human skin cancer because it can spread throughout the body.

Scientists are using powerful supercomputers to uncover the mechanism that activates cell mutations found in about 50 percent of melanomas. The scientists say they're hopeful their study can help lead to a better understanding of skin cancer and to the design of better drugs.

In 2002, scientists found a link between skin cancer and mutations of B-Raf (Rapidly Accelerated Fibrosarcoma) kinase, a protein that's part of the signal chain that starts outside the cell and goes inside to direct cell growth. This signal pathway, called the Ras/Raf/Mek/Erk kinase pathway, is important for cancer research, which seeks to understand out of control cell growth. According to the study, about 50 percent of melanomas have a specific single mutation on B-Raf, known as the valine 600 residue to glutamate (V600E).

B-Raf V600E thus became an important drug target, and specific inhibitors of the mutant were developed in the following years. The drugs inhibited the mutant, but something strange happened. Paradoxically, quieting the mutant had a down side. It activated the un-mutated, wild-type B-Raf protein kinases, which again triggered melanoma.

"With this background, we worked on studying the structure of this important protein, B-Raf," said  the co-author of the study in the journal Science that determined the structure of the complex of proteins that make up B-Raf and also found how the paradoxical B-Raf activation happens.

"We aimed to study the more native-like state of the protein to understand how it's regulated in the cells, because most of the studies have been focused on the isolated kinase domain and how the drugs bind to the kinase domain." said the co-author.

The full-length B-Raf protein is made of several domains linked by disordered regions, something too unwieldy for scientists to yet image. The authors technique was to use intein chemistry to make smaller fragments, then stitch them up to get the full structure.

"As a result, we obtained an active form of the full-length B-Raf dimer called B-Raf co-purified with 14-3-3 dimer, a scaffolding protein bound to the phosphorylated B-Raf C-terminal tail," the co-author said.

The group used cryo-electron microscopy (cryo-EM) to determine the structure of B-Raf 14-3-3 complex, basically cryogenically freezing the protein complex, which kept it in a chemically-active, near-natural environment. Next they flashed it with electron beams to obtain thousands of 'freeze frames.' They sifted out background noise and reconstructed three-dimensional density maps that showed previously unknown details in the shape of the molecule. And for proteins, form follows function.

The co-author explained that the structure revealed an asymmetric organization of the complex, formed by two sets of internally symmetrical dimers, or pairs of bonded molecules. "We propose that this unexpected arrangement enables asymmetric activation of the B-Raf dimer, which is a mechanism that provides an explanation of the origin of the paradoxical activation of B-Raf by small molecule inhibitors," the co-author said.

Detailed analysis of the asymmetrical B-Raf 14-3-3 complex structure showed another unexpected structural feature, described as the distal tail segment, DTS for short, of one B-Raf molecules. The co-author said the tail of one is bound to the active site of the other, blocking its activity by competing with ATP binding. The blocked B-Raf molecule is stabilized in the active conformation. "We interpreted this structure that this blocked B-Raf molecule functions as an activator and stabilizes the other B-Raf receiver through the dimer interface," the co-author said.

Curiously enough, the authors compare the B-Raf dimer to the Chinese yin-yang circular symbol of interconnected opposites joined at the tail. "From looking at the subject, it's very clear that one is not capable of phosphorylating the downstream molecule, which is necessary for cell growth. The other molecule is clearly the one to do the job. In this set of two molecules, we clearly see one is doing the supporting job, and the other one is doing the actual work. It really does look like Yin and Yang in this B-Raf 14-3-3 complex we solved," the co-author said.

Looks, though, can be deceiving. Scientists used computer simulations to help verify that they were really onto something. "We ran molecular dynamics simulations of this complex of the B-Raf dimer bound to a 14-3-3 dimer to test the stability of the asymmetric conformation," said study’s another co-author. "We didn't know why the conformation was asymmetric, or what role it played in maintaining the active state of the enzyme," the other co-author said.

They started the simulations using the structure that was solved by cryo-EM, with the DTS segment running from one kinase into the active site of the other. Then they ran a second set of simulations with the DTS segment removed.

"What we found was that in the system without the distal tail segment, the entire complex is not stable," thr other co-author explained. "The kinase domains move with respect to the scaffolding, the 14-3-3 dimer. In one of our simulations, the dimer state of B-Raf itself, which experiments have shown is necessary to maintain the active state of this kinase, it fell apart, indicating that this distal tail segment, DTS, is necessary to actually maintain this complex in this asymmetric conformation, which in turn is necessary to maintain the kinase dimer in the stable asymmetric dimer active state."

One of the main results of the study was finding the mechanism of action that switches on the B-Raf kinase complex of two B-Raf kinases and two 14-3-3 scaffolding proteins, where on B-Raf kinase is the activator, and the other is the receiver.

"The tail of the receiver molecule is inside the active site of the activator, so the activator cannot work as an enzyme," the co-author said. "Instead, the activator molecule stabilizes the active conformation of the receiver molecule. The 14-3-3 scaffold protein facilitates this arrangement, so that the tail insertion only happens to one kinase molecule. We hypothesize that when there is no 14-3-3 binding, both kinases can be blocked by the insertion of the DTS, but this needs to be tested."

"The structure that was resolved in this paper is part of a large, multi-domain system," the other co-author explained. "We don't know what this complete protein looks like. We don't see it in the structure. We don't know what its dynamics look like, and how all these other parts of the protein play a role in maintaining the active state, or converting it from the inactive state to the active state."

https://www.tacc.utexas.edu/-/skin-cancer-mystery-revealed-in-yin-and-yang-protein

https://science.sciencemag.org/content/366/6461/109

Edited December 19th 2019