CB1 Protein Structure

by Andrew Zale

Not to stir the proverbial pot with a controversial topic, but while weeding through recent science literature I encountered an interesting article seeking to clear the smoke surrounding some biological symptoms. The bloodshot eyes. The lethargic drowsiness. The “munchies.” The symptoms of marijuana use have been well-documented, if anything due to its prevalence: a 2017 letter from the University of Pittsburgh’s Division of Student Affairs estimated that 4.6% of Pitt students consume marijuana regularly. Prior to this summer, however, the molecule responsible for the high that marijuana users experience—a protein known as cannabinoid receptor type 1 (CB1) —was not well understood.

Chinese researchers, in collaboration with scientists from Northeastern University, The Scripps Research Institute and the University of Southern California, published a crystal structure, a 3-D molecular model, of CB1 bound to substrates that activate and inactivate the “high” response.

What is CB1?

CB1 is a protein receptor found primarily in the central nervous system, which includes the brain and the spinal cord. The only other cannabinoid receptor that has been discovered is CB2, which is localized in the immune system and brain and triggers an effect on the immune system’s leukocytes. Since CB1 is located throughout the nervous system, substrates such as THC, the psychoactive drug in marijuana, can bind and activate CB1, causing the sensational high. Without substances like THC in the body, CB1’s binding pocket remains unfilled and thus causes the protein’s inactivation, preventing the high response that occurs when you smoke.

Understanding the shape of CB1 is crucial. Arguably the most fundamental physiological concept states that structure determines function: understanding the structure of a receptor protein helps explain how it is activated. Receptor proteins have binding pockets in which small molecules like THC can fit. Much like the “square peg in a round hole” analogy, molecules that do not have the proper shape cannot fit into and activate these receptor proteins. This gives the receptors their unique specificity. If CB1’s receptor did not have a specific shape, many small molecules could fit into the binding site and activate the protein. This would result in a high even when you haven’t smoked THC. In addition to the shape specificity, the molecule must have the proper conformation: just like a key can only enter a keyhole in one direction, a molecule may be able to recognize a receptor protein but will not be able to activate it because its conformation when the protein and ligand interact is incorrect. The specificity of CB1 explains why certain cannabinoids like THC can get you high, but certain cannabinoids like cannabidiol, which is also found in cannabis alongside THC, do not: CB1 cannot bind cannabidiol, as demonstrated in a 2008 paper from the British Journal of Pharmacology.

How was CB1’s Structure Determined?

Researchers used X-ray crystallography in order to determine CB1’s structure in the presence of certain substrates. The scientists produced the protein in human cells and purified it in vitro for X-ray crystallography. Following protein purification, the protein was placed in different chemical environments until adequate crystals were formed. The protein of interest must be purified to essentially 100% purity because contaminants within the protein solution will prevent the formation of crystals. Once the crystal-forming environment was determined by scientists, small modifications were made until large, evenly shaped crystals were formed.

Protein crystallization is very difficult because not only does the protein need to be pure of contaminants, but it must also be rigid: flexible regions of protein will not crystalize. Therefore, scientists may co-crystallize a protein with its substrate in order to ensure that the protein is found only in one conformation: tightly bound to the substrate. The bound conformation is less flexible and therefore increases the likelihood of protein crystallization. Two chemically synthesized molecules used as model substrates for THC, AM11542 and AM841, named after Dr. Alexandros Makriyannis of Northeastern University and an author on this THC crystal structure journal article, were crystallized with CB1. Both AM11542 and AM841 potently bind CB1 much more strongly than THC, which creates a stabilizing interaction between the AM11542 or AM841 and CB1, allowing CB1 to be more easily crystalized.

Dr. Tian Hua of ShanghaiTech University and first author on the paper explained in an interview with Analytical Cannabis “Crystal structures of [CB1] have been challenging to obtain due to their increased conformational flexibility and resulting instability.”

“We are thrilled to see the agonists AM11542 and AM841 could significantly stabilize the conformation of the receptor and lead to the structure determination. [AM11542 and AM841 bound CB1] complexes reveal significant conformational changes in the overall structure” elaborated Dr. Raymond Stevens of ShanghaiTech University in the same interview.

The obtained crystals were then one-by-one placed into a machine that shot X-rays at the crystal; based on the crystal’s definitive structure, the X-rays were diffracted in a specific pattern unique to its shape. After repeated measurements, the machine’s artificial intelligence determined a structure that was refined by the researchers.

What did the Structure Show?

CB1 has eight hotdog-like helices, seven of which are embedded in the cell membrane, connecting the inside and outside of the cell, and one helix completely within the cell. The seven hotdog-like helices form a pocket that allow certain molecules to fit. These structures, known as alpha-helices are like coiled wires. These coiled wires are then folded in such a way that creates a specific, 3-D structure that includes a THC-binding pocket.

According to Makriyannis’ research, when molecules like THC, which activates the receptor, were in the binding pocket, the protein changed its conformation to reduce the pocket size by 53%. THC never enters the cell, so its message is transmitted across the cell membrane via a signal transduction pathway. Essentially, once THC binds to CB1 on the outside of the cell, another protein bound to CB1 inside the cell is activated, which can pass the message on to other proteins and molecules, eventually eliciting a response. The decrease in the size of the binding pocket caused an increase in the available area for intracellular proteins to bind to another region of CB1. When THC binds to CB1’s binding pocket, the protein’s shape changes, allowing other proteins to bind to CB1 and receive the message due to this conformational change. This signal transduction pathway is similar to a baton race where the message gets passed from molecule to molecule, and THC binding to CB1 changes CB1’s conformation to pass the baton to other proteins. Scientists determined that CB1’s binding pocket was malleable, meaning that the pocket is more like playdough than a keyhole with regards to the molecules that can bind to it.

Where do We Go from Here?

As described above, the binding pocket of CB1 is relatively flexible, which may allow scientists to synthesize molecules resembling THC that have similar effects. The structural understanding of what can fit into the binding pocket and the duration with which molecules will stay in the pocket may lead to safer synthetic marijuana production. Synthetic THC-analogs, like K2 and spice, have shown to be addictive whereas THC does not, as described in a 2014 Life Sciences paper. The dangers of K2 and spice relative to THC remain rather mysterious and unknown, but the crystal structure and how long each drug can stay bound in CB1’s binding pocket may elucidate why some drugs are more dangerous than others. Conversely, drug design has also sought to produce substances that antagonize and shut down CB1 rather than induce more potent highs. Drugs have been synthesized by Makriyannis that can turn off the CB1 receptor entirely.

In an interview with Wired, Makriyannis explains “We want to make compounds that will modify the receptor differently, so we can make better drugs.”

In an age where the medicinal applications of marijuana are questioned and investigated, understanding possible substrates for CB1 will allow for more effective drug research and a better comprehension on the molecular level of why people get “high.”