Freezing a Bullet (+)

X-Ray Vision, an article in our Spring 2015 issue, examined the central role Caltech has played in developing a powerful technique for revealing the molecular machinery of life. In May, chemist André Hoelz, who was featured in the article, published a new paper describing how he used the technique to reveal how protein-synthesizing cellular machines are built.

Crystal structure of the assembly chaperone of ribosomal protein L4 (Acl4) that picks up a newly synthesized ribosomal protein when it emerges from the ribosome in the cytoplasm, protects it from the degradation machinery, and delivers it to the assembly site of new ribosomes in the nucleus.   Credit: Ferdinand Huber/Caltech
Crystal structure of the assembly chaperone of ribosomal protein L4 (Acl4) that picks up a newly synthesized ribosomal protein when it emerges from the ribosome in the cytoplasm, protects it from the degradation machinery, and delivers it to the assembly site of new ribosomes in the nucleus.
Credit: Ferdinand Huber/Caltech

Ribosomes are vital to the function of all living cells. Using the genetic information from RNA, these large molecular complexes build proteins by linking amino acids together in a specific order. Scientists have known for more than half a century that these cellular machines are themselves made up of about 80 different proteins, called ribosomal proteins, along with several RNA molecules and that these components are added in a particular sequence to construct new ribosomes, but no one has known the mechanism that controls that process.

Now researchers from Caltech and Heidelberg University have combined their expertise to track a ribosomal protein in yeast all the way from its synthesis in the cytoplasm, the cellular compartment surrounding the nucleus of a cell, to its incorporation into a developing ribosome within the nucleus. In so doing, they have identified a new chaperone protein, known as Acl4, that ushers a specific ribosomal protein through the construction process and a new regulatory mechanism that likely occurs in all eukaryotic cells.

The results, described in a paper that appears online in the journal Molecular Cell, also suggest an approach for making new antifungal agents.

The work was completed in the labs of André Hoelz, assistant professor of chemistry at Caltech, and Ed Hurt, director of the Heidelberg University Biochemistry Center (BZH).

“We now understand how this chaperone, Acl4, works with its ribosomal protein with great precision,” says Hoelz. “Seeing that is kind of like being able to freeze a bullet whizzing through the air and turn it around and analyze it in all dimensions to see exactly what it looks like.”

That is because the entire ribosome assembly process—including the synthesis of new ribosomal proteins by ribosomes in the cytoplasm, the transfer of those proteins into the nucleus, their incorporation into a developing ribosome, and the completed ribosome’s export back out of the nucleus into the cytoplasm—happens in the tens of minutes timescale. So quickly that more than a million ribosomes are produced per day in mammalian cells to allow for turnover and cell division. Therefore, being able to follow a ribosomal protein through that process is not a simple task.

Hurt and his team in Germany have developed a new technique to capture the state of a ribosomal protein shortly after it is synthesized. When they “stopped” this particular flying bullet, an important ribosomal protein known as L4, they found that its was bound to Acl4.

Hoelz’s group at Caltech then used X-ray crystallography to obtain an atomic snapshot of Acl4 and further biochemical interaction studies to establish how Acl4 recognizes and protects L4. They found that Acl4 attaches to L4 (having a high affinity for only that ribosomal protein) as it emerges from the ribosome that produced it, akin to a hand gripping a baseball. Thereby the chaperone ensures that the ribosomal protein is protected from machinery in the cell that would otherwise destroy it and ushers the L4 molecule through the sole gateway between the nucleus and cytoplasm, called the nuclear pore complex, to the site in the nucleus where new ribosomes are constructed.

“The ribosomal protein together with its chaperone basically travel through the nucleus and screen their surroundings until they find an assembling ribosome that is at exactly the right stage for the ribosomal protein to be incorporated,” explains Ferdinand Huber, a graduate student in Hoelz’s group and one of the first authors on the paper. “Once found, the chaperone lets the ribosomal protein go and gets recycled to go pick up another protein.”

The researchers say that Acl4 is just one example from a whole family of chaperone proteins that likely work in this same fashion.

Hoelz adds that if this process does not work properly, ribosomes and proteins cannot be made. Some diseases (including aggressive leukemia subtypes) are associated with malfunctions in this process.

“It is likely that human cells also contain a dedicated assembly chaperone for L4. However, we are certain that it has a distinct atomic structure, which might allow us to develop new antifungal agents,” Hoelz says. “By preventing the chaperone from interacting with its partner, you could keep the cell from making new ribosomes. You could potentially weaken the organism to the point where the immune system could then clear the infection. This is a completely new approach.”

Co-first authors on the paper, “Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension,” are Huber and Philipp Stelter of Heidelberg University. Additional authors include Ruth Kunze and Dirk Flemming also from Heidelberg University. The work was supported by the Boehringer Ingelheim Fonds, the V Foundation for Cancer Research, the Edward Mallinckrodt, Jr. Foundation, the Sidney Kimmel Foundation for Cancer Research, and the German Research Foundation (DFG).

—Kimm Fesenmaier

A Lifetime of Crystallography (+)

Chemist Dick Marsh, a Techer through and through, has witnessed the evolution of X-ray crystallography (see “X-Ray Vision“) since the era of Linus Pauling.

Dick Marsh in 2012 Photo courtesy of the American Crystallographic Association
Dick Marsh in 2012

He studied applied chemistry at Caltech, receiving his undergraduate degree in 1943. He then joined the Navy, where his job was to degauss ships so they wouldn’t trigger magnetic mines.

After the war, he enrolled in graduate school at Tulane University, but when he tried to sign up, most of the classes were already filled. The registrar found one available class—at Sophie Newcomb College, the women’s college next door. The class was on X-ray crystallography, the study of crystals and their structure using X-ray diffraction.

As he wrote later in an essay for the American Crystallographic Association, he had never heard of X-ray crystallography. But the class changed his life, as he credits his instructor, Rose Mooney, for inspiring him to become a crystallographer.

(As a side note, according to Marsh’s essay, Mooney had been accepted to Caltech’s graduate program a few years prior. She didn’t know Caltech didn’t allow women at the time and the university didn’t know “R.C.L. Mooney” was a woman. Marsh wrote that Linus Pauling would help arrange a research assistantship for her and help transfer her to the University of Chicago, where she got her PhD.)

Marsh later transferred to UCLA for his PhD and returned to Caltech as a postdoc in 1950, focusing on the structures of smaller molecules. It was the heyday of structural chemistry. People from all over the world were flocking to Pasadena to work with Pauling, Marsh says.

Dick Marsh and Linus Pauling at Pauling’s 85th birthday celebration.

The first paper he published at Caltech was with Pauling, in which they used crystallography to find the structure of a molecule called chlorine hydrate. Pauling was extremely cogent and articulate, Marsh recalls. After solving a molecular structure, Marsh would show the results to Pauling, who would take a look and speak into a recording device, dictating an entire paper from start to finish for his secretary to type out.

Through his more than six-decade career at Caltech, Marsh has seen how computers have revolutionized the field. “When I started, I had a slide rule and a pencil,” he says. Now, in his role as senior research associate in chemistry, emeritus, he still goes to work every day. Solving chemical structures is a great puzzle, he says, and it’s the joy of cracking these riddles that keeps him going. Asked if he has any favorite discoveries after all these years, he can’t choose. “They’ve all been fun,” he says.

–Marcus Woo

Photos courtesy of the American Crystallographic Association


X-Ray Vision

In the past, the calculations needed 
to interpret X-ray diffraction patterns and solve molecular structures were the most time-consuming part of crystallography. Now, X-ray technology is constantly improving, with better detectors and with beams that can be controlled with greater and greater precision, allowing researchers to analyze bigger and more complex structures. Computers now take less than a second to do the math that once took weeks 
by hand. These advances have made crystallography nearly limitless in its potential, Andre Hoelz says. Researchers can study as complex a system as they wish. “We pick a project and we make it happen,” he says.

To aid in their experiments, Caltech researchers have access to the Molecular Observatory, which includes an automated X-ray beam line at the Stanford Synchrotron Radiation Laboratory. The campus also has its own in-house macromolecular X-ray crystallography facility. The Macromolecular Crystallization Laboratory in the Beckman Institute provides automated and robotic facilities (yes, there are actual robots) to help researchers prepare their samples efficiently.

Such automated methods allow researchers to do experiments with much less sample material than before, and thus to solve more challenging problems. According to Bil Clemons, in the roughly 15 years since he started doing crystallography the amount of sample protein needed has dropped 
by about 20-fold. Still, preparing
 and crystallizing samples is laborious, and is the hardest part of crystallography today.

Click here for the full feature story…

 Header Photo by Thomas Spatzal and Doug Rees