The baker’s son

The phone rang at 2:20 a.m. It was early October 2012.

Brian Kobilka didn’t get up to answer it. The M.D. and researcher figured it was a wrong number.

A few minutes later, the phone rang again.

“I answered the phone and it was a woman with a Swedish accent, telling me that she would like me to speak to the chair of the chemistry committee,” Kobilka said. “It was not the scenario that I would have envisioned for a prank call.”

The chairman told him the news. The Nobel Committee chose Kobilka and his mentor, Robert Lefkowitz, M.D., as the 2012 co-recipients of the Nobel Prize for Chemistry.

“He actually sent congratulations from a Swedish scientist that I know, and nobody would have known to do that.”

In less than 15 seconds, it became real for Kobilka.

“At first I was just in shock. I didn't have much time to be skeptical. It was believable because the process didn't go as I would expected if it were a hoax.”

Kobilka, born in 1955, was a professor at Stanford Medical School when he got the news. He went to work that day, but admittedly didn’t get much done.

Kobilka and Lefkowitz, along with a large team of collaborators, had described the structure and workings of G protein-coupled receptors (GPCRs). These are part of a large family of human proteins, which are the target of almost half of pharmaceuticals today.

Our body’s functions require extensive communication between different parts of the body. That communication occurs electrically through nerves, and at the end of nerves, a release of small molecules called neurotransmitters.

The brain regulates cardiovascular function through a special network called the autonomic nervous system. Neurotransmitters released from these nerve endings in the heart can make the heart speed up or slow down. The neurotransmitters relay information to heart muscle cells by activating special GPCRs.

Similarly, when a hormone is released into the blood it, it is distributed throughout the body and it will find a receptor that is responsive to that hormone.

“So for example, if you've taken too much insulin, your blood sugar drops, Kobilka explained. “The pancreas secretes a hormone called glucagon, which is recognized by a specific GPCR on the surface of liver cells, and the liver starts releasing glucose. Thus, GPCRs relay information from hormones and neurotransmitters from the outside of the cell to the inside of the cell, typically through proteins called G proteins.”

Therefore, a receptor receives information from a hormone or neurotransmitter that causes structural changes in the receptor, which then allows it to activate G proteins, which change the behavior of the cell.

“My contribution was determining the three-dimensional structure of these GPCRs in both inactive and active states, and determining the structure of a GPCR in complex with its G protein,” Kobilka said.

Now researchers can now use computational methods to screen for drugs for G protein-coupled receptors for which structures are available.

“We developed protein engineering strategies for obtaining GPCR crystal structures,” he said. “So after our initial crystal structures, many others have been able to crystallize GPCRs in both inactive and active states. Now, many of these structures are available, and computer-based drug screening is now very much a viable approach for this family of receptors.”

Researchers have identified nearly 800 different GPCRs, making them one of the largest families of human proteins. These proteins regulate nearly every bodily process, including the workings of our brains and the beating of our hearts.

The medications that target these receptors include familiar drugs like Zyprexa, which practitioners use to treat schizophrenia; Clarinex, an antihistamine; and Zantac, which is used for stomach ulcers and gastro-esophageal reflux disease.

GPCRs are also involved in some kinds of drug addictions, such as addiction to morphine and other opiates. In fact, Kobilka co-authored a 2016 study that used a structure-based approach to identify a new opioid drug. In animal studies, this drug possesses pain-inhibiting qualities without the potentially lethal respiratory suppression.

Kobilka, a self-confessed introvert, speaks softly without a hint of pretention. He projects a boyish shyness. But his speech quickens when he’s talking about his research, or his long list of collaborators. That’s his passion.

At about 6 foot 1 inch, he’s tall and lanky with an endurance athlete’s build. In fact, he’s been a hard-core runner and bicyclist, once riding the mountains of the Pyrenees with his son during the Tour de France. These days he settles for a bicycle mounted on a trainer stand for indoor workouts.

Kobilka’s office is absent of any tributes or displays of his Nobel achievement.

The walls of his compact office include a photo of his grandchildren and small piece of framed artwork. A large whiteboard takes up much of one wall, partially covered with equations and graphs in colored markers. But dominating the board is a drawing in green marker by his granddaughters, perhaps covering up the scientist’s next breakthrough. Kobilka is unfazed – this in one low-key guy.

He does have one memento that drapes over his chair – a commemorative championship University of Minnesota-Duluth burgundy hockey jersey, signed around the lower band by his teachers from when he was a student there.

A rectangular table in his office serves as a workspace for his wife, Tong Sun, M.D., a practicing internist, who helps out in the lab part time. An associate sits in an outer office.

Kobilka grew up in Little Falls, Minnesota, with a population of 7,500. It sits alongside the Mississippi River.

His parents owned a bakery, and Kobilka and his sister worked there from the age of 13. The experience shaped his personality.

The bakery was relatively large for a small town, with a half-dozen full time employees and several part time high-school students. It gave him the chance to watch how his father interacted with others.

“I saw how he was able to get the best out of everybody through personal interactions that were supportive,” Kobilka said. “He would help people financially if they needed it. I only learned about this after the fact from my mother. My dad was generous and treated other people with respect and kindness. If I incorporated those traits into my own personality, I got it from seeing how my father managed his bakery.”

Kobilka dabbled in research as a biology undergrad at Duluth, but applied to medical schools, along with a couple of backup applications for research. He ended at the Yale School of Medicine to pursue a medical degree.

Culture shock for the mid-westerner accompanied his move to New Haven, Connecticut. The school is located in the inner city, where crime and poverty were pervasive. Research was part of the curriculum, and his first project was a study of dengue fever in a lab in Malaysia.

He and Tong Sun, who he met at the University of Minnesota, Duluth, married in 1978, after his first year at Yale.

His tuition assistance under the Public Health Service program required him to work in a medically underserved community for three years following his residency training after graduation. That took basic research off the table. Kobilka chose a residency in internal medicine in St. Louis.

But a loss in funding for Public Health Service programs allowed Kobilka to pay back his medical school scholarship by working in an academic hospital for three years. That gave him the opportunity to explore basic research.

He became interested in intensive care medicine, where patients were usually unstable. Patients required urgent intervention, often with medications acting on G protein coupled receptors. That included adrenergic (nerve cell) and muscarinic receptors to regulate their heart rate and blood pressure, along with opioid receptors to control pain.

He applied for and was accepted into a cardiology fellowship at Duke University and gained access to Robert Lefkowitz’s lab. At the time, the lab was known for its groundbreaking research on adrenergic receptors. That wed the areas of basic research with cardiovascular and intensive care medicine central to Kobilka’s interests.

He moved to a drafty, two-story apartment close to the lab in Durham, North Carolina, often running to and from the lab while leaving the family car for Tong Sun and their children. The roundtrip was several miles, depending on his route.

That was only part of his transition.

“On joining the Lefkowitz lab, I became the least experienced person in a group of very talented young scientists consisting of predominantly postdoctoral fellows and a few graduate students,” he wrote. “I was not familiar with any of the techniques being used and had yet to familiarize myself with the literature leading up to work being done in the Lefkowitz lab at that time. My colleagues were very friendly, but also very busy with their own projects and I really didn’t know where to begin.”

He spent time at Merck Laboratories north of Philadelphia learning molecular biology from Richard Dixon, where he learned how to prepare and screen cDNA libraries. Returning to Duke, Kobilka set up a molecular biology lab within the Lefkowitz lab.

The initial goal was to screen libraries prepared by Dixon. Many initial results turned out to be non-specific interactions with various probes, accompanied by the euphoria and discouragement typical of basic research.

The group came to believe that after about a year of failure, β2ARs (the β2 adrenergic receptors) were present at such low levels in most cells that the researchers might not be able to isolate a clone from a cDNA (complimentary DNA synthesized from single-stranded RNA) library.

Ultimately, groups at Merck and Duke were able to isolate a genomic clone that contained the full coding sequence with no introns – a DNA or RNA molecule which does not code for proteins and interrupts the sequence of genes.

That led to the cloning of several other adrenergic receptor genes as well as an orphan receptor. That receptor turned out to be a member of the serotonin (neurotransmitter) receptor family. The initial clones had a common, seven transmembrane architecture just like rhodopsin, a G protein coupled receptor that specialized in the detection of light.

Yet, the studies did not show how the receptor worked in molecular detail. Kobilka began to think about obtaining a crystal structure.

In the meantime, Professor Richard Tsien, Ph.D. just moved from Yale to Stanford University. He was building a new Department of Molecular and Cellular Physiology in Palo Alto. Tsien was a popular lecturer and visionary during Kobilka’s med student days at Yale, and Kobilka took a position in the new department.

The increase in housing costs and Tong Sun’s entry into medical school forced Kobilka to take 48 hour shifts as an emergency room physician at a nearby hospital a couple of times a month. But with his wife a full-time student, it also gave him a chance to spend more time with his son and daughter as their primary care giver.

Emergency medicine, though, with its instant gratification, doesn’t seem to share the same rewards as the slow progress of basic research.

Kobilka disagreed.

“With emergency medicine or intensive care medicine, you get a lot of feedback, so you can, in a short period of time see the result of your therapeutic intervention,” he said. “So you can make judgements and you can see patients get better.”

“In terms of research, if you're doing something really challenging, then you can't expect instant gratification, but each little incremental advance you make, like an experiment, may be part of just a big story. That experiment can happen over short period and you can see something interesting happen. And that will give you an essential piece of information to move forward. So there are small advances during a prolonged project that can be exciting.”

Kobilka focused on two questions in the lab:

1. Determining the physiologic role of specific adrenergic receptor subtypes.
2. Understanding the structure and mechanism of activation of the β2AR.

Kobilka used knockout mice to achieve the first goal.

A knockout mouse is a laboratory mouse where researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA.

Along with his colleague Greg Barsh, he created strains of knockout mice for five of the nine adrenergic receptor genes, and they were able to assign their roles in cardiovascular function and behavior.

To achieve the second goal, he had to be able to make large quantities of receptor protein.

“By 1993,” he said, “we were able to express and purify sufficient quantities of functional β2AR in insect cells to begin using fluorescence spectroscopy, one of the most sensitive biophysical techniques, to investigate receptor structure. We labeled purified β2 adrenergic receptor (β2AR) with small, environmentally sensitive fluorescent probes, most often attaching them to a single reactive cysteine introduced into a specific domain.”

“Using this approach, we were able to observe ligand-induced conformational changes in real time. These relatively simple fluorescence experiments provided important insights into the dynamic character of the β2AR that would ultimately guide our strategies for crystallizing the β2AR and the β2AR-Gs complex.”

The crystal structure of a protein provides a model or template for the binding pocket of the hormone and neurotransmitter. A computer can store a virtual library of compounds. Researchers can test many drugs and molecules to see if they bind, and then the computer can rank these according to ones that bind.

Through incremental achievements, Kobilka’s group was able to produce enough β2AR to start crystallography trials. Kobilka’s Stanford colleague, Harvard trained Ph.D. Bill Weis, taught him how to set up the trials.

In 2004, after many false starts, the team achieved the first crystals of the β2AR. Kobilka called it “an important milestone.”

Dan Rosenbaum and Søren Rasmussen, both postdoctoral fellows aimed to crystalize the β2AR. They subsequently obtained a 3.4Å (ångström, each equaling 0.1 nm a unit) of the β2AR-Fab complex.

“This was our first look at the three dimensional structure of the β2AR, but a higher resolution structure would soon follow,” Kobilka said.

And it did, through a collaboration with Scripps.

These first β2AR structures represented inactive states. Yet, the team’s goal was to understand how agonist binding leads to G protein activation.

Through an intense collaboration using a variety of methods to study activation of the G protein Gs by the β2AR, Kobilka and his colleagues accumulated the reagents and expertise to stabilize and crystallize the β2AR-Gs complex.

The team published the β2AR-Gs crystal structure in 2011 together with two companion studies to characterize the molecule’s dynamic aspects.

“These combined studies provided unprecedented insights into GPCR signaling at a molecular level,” he said.

Like most researchers, Kobilka didn’t experience a single monumental breakthrough moment. It was more of a process, a trial and error approach.

“When an experiment failed, we tried to figure out why it failed,” he said. “Because often you learn something from that. It helps you plan the next experiment.”

A certain attitude accompanies basic research, since it's so intensive and slow to develop.

“You've got these important goals, like identifying new or better drugs challenging diseases. But the steps are so incremental.”

It wasn’t necessarily the desire to develop a better drug that motivated Kobilka.

“It's just a level of curiosity, that you really want to know how something works,” he said. In many cases you can't keep using the same method over and over. You have to be willing to try methods that you're not necessarily an expert in. I've always liked the fact that I've had to learn a lot new methods and techniques to be able to solve these problems.”

Spectroscopy was one of those.

“You don’t learn those things in medical school, “he said. “So you find colleagues who become collaborators and are generous enough with their time to teach you.”

Kobilka’s team tried a variety of experiments to understand how receptors engaged G proteins. They used steady state fluorescence spectroscopy, which taught them how to try to stabilize a receptor for structural studies.

Kobilka uses a Fluorolog3/TCSPC, (Time-Correlated Single-Photon Counting) spectrofluorometer in his lab. The modular instrument can look at molecular, chemical and electronic properties down to the nanoscopic level.

“From the very beginning of my time at Stanford, I've wanted to learn about what GPCRs look like in three dimensions,” he said. “It took a long time for us to be able to produce enough protein to start crystallography. But early on, we could produce enough protein to begin to look at lower resolution readouts of protein structure. And fluorescence was ideal because you don't need much protein. If you can label the protein with a small fluorophore, you can learn about how it changes conformation under different conditions.”

The team directed fluorescent probes to the end of a transmembrane segment that spans the entirety of the cell membrane. They could see large structural changes in going from say, an antagonist to an agonist and then even additional changes going from an agonist to an agonist plus G protein.

“By having that kind of information, those studies taught us a lot about how these proteins were more dynamic than we thought.”

The Nobel committee presents the Nobel Prizes at ceremonies on December 10 of each year, the anniversary of Alfred Nobel's death.

Kobilka and his fellow Nobel Laureates arrived in Stockholm, Sweden in December 2012, a few days before the awards ceremony. A week of festivities and parties in the city complements the event.

“They’re really welcoming, and make you really feel special,” he said.

Kobilka accompanied his fellow 2012 laureates in physics, economics, medicine, and literature. The city treated them like celebrities, even heroes. Yet, despite all the accolades, his Midwestern humility remains.

“There’s a lot of luck that goes into winning, because there's so many deserving scientists,” he said. “The timing has to be right. They have to refine the right combination of people to share it. And I had so many colleagues who contributed to it.”

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