A close look at one protein shows how it moves molecular passengers into cells in the kidneys, brain and elsewhere.

The protein LRP2 is part of a delivery service, catching certain molecules outside a cell and ferrying them in. Now, 3-D maps of LRP2 reveal the protein’s structure and how it captures and releases molecules, researchers report February 6 in Cell. The protein adopts a more open shape, like a net, at the near-neutral pH outside cells. But in the acidic environment inside cells, the protein crumples to drop off any passengers.
The shape of LRP2’s structure — and how it enables so many functions — has stumped scientists for decades. The protein helps the kidneys and brain filter out toxic substances, and it operates in other places too, like the lungs and inner ears. When the protein doesn’t function properly, a host of health conditions can occur, including chronic kidney disease and Donnai-Barrow syndrome, a genetic disorder that affects the kidneys and brain.

The various conditions associated with LRP2 dysfunction come from the protein’s numerous responsibilities — it binds to more than 75 different molecules. That’s a huge amount for one protein, earning it the nickname “molecular flypaper,” says nephrologist Jonathan Barasch of Columbia University.

Typically, LRP2 sits at a cell membrane’s surface, waiting to snag a molecule passing by. After the protein binds to a molecule, the cell engulfs the part of its surface containing the protein, forming an internal bubble called an endosome. LRP2 then releases the molecule inside the cell, and the endosome carries the protein back to the surface.

To understand this shuttle system, Barasch and colleagues collected LRP2 from 500 mouse kidneys. The researchers put some of the protein in a solution at the extracellular pH of 7.5, and some in an endosome-mimicking solution at pH 5.2. Using a cryo-electron microscope, they captured images of the proteins and then stitched the images together in a computer, rendering 3-D maps of the protein at both open and closed formations.
The researchers suggest that charged calcium atoms hold the protein open at extracellular pH. But as pH drops due to hydrogen ions flowing into the endosome, the hydrogen ions displace the calcium ions, causing the protein to contract.

In the early morning of February 6, a devastating magnitude 7.8 earthquake struck southern Turkey, near the border with Syria. Numerous aftershocks followed, the strongest nearly rivaling the power of the main quake, at magnitude 7.5. By evening, the death toll had climbed to more than 3,700 across both countries, according to Reuters, and was expected to continue to rise.

Most of Turkey sits on a small tectonic plate that is sandwiched between two slowly colliding behemoths: the vast Eurasian Plate to the north and the Arabian Plate to the south. As those two plates push together, Turkey is being squeezed out sideways, like a watermelon seed snapped between two fingers, says seismologist Susan Hough of the U.S. Geological Survey.
The entire country is hemmed in by strike-slip, or sideways-sliding, fault zones: the North Anatolian Fault that runs roughly parallel to the Black Sea, and the East Anatolian Fault, near the border with Syria. As a result, Turkey is highly seismically active. Even so, Monday’s quake, which occurred on the East Anatolian Fault, was the strongest to strike the region since 1939, when a magnitude 7.8 quake killed 30,000 people.

Science News talked with Hough, who is based in Pasadena, Calif., about the quake, its aftershocks and building codes. The conversation has been edited for length and clarity.

SN: You say on Twitter that this was a powerful quake for a strike-slip fault. Can you explain?

Hough: The world has seen bigger earthquakes. Subduction zones generate the biggest earthquakes, as much as magnitude 9 (SN: 1/13/21). But quakes close to magnitude 8 are not common on strike-slip faults. But because they’re on land and tend to be shallow, you can get severe … shaking close to the fault that’s moving.

SN: Some of the aftershocks were very strong, at magnitudes 7.5 and 6.7. Is that unusual?

Hough: As with a lot of things, there’s what’s expected on average, and there’s what’s possible. On average, the largest aftershocks are a full unit smaller than the main shock. But that’s just average; for any individual main shock, the largest aftershock can have a lot of variability.

The other thing people noted was the distance [between the main shock and some aftershocks over a hundred kilometers away]. Aftershock as a term isn’t precise. What is an aftershock isn’t something that seismologists are always clear on. The fault that produced the main shock is 200 kilometers long, and that’s going to change the stress in a lot of areas. Mostly it releases stress, but it does increase stress in some areas. So you can get aftershocks along that fault, but also some distance away. It’s a little bit unusual, but not unheard of.

SN: People have wondered whether Monday’s magnitude 3 earthquake near Buffalo, N.Y., might be related.

Hough: A magnitude 7.8 quake generates [seismic] waves that you can record all around Earth, so it’s technically disrupting every point on Earth. So it’s not a completely outlandish idea, but it’s statistically exceedingly unlikely. Maybe if a seismic wave passed through a fault that was just ready to go in just the right way, it’s possible.

An interesting [and completely separate] idea is that you might get earthquakes around the perimeter of the Great Lakes [such as near Buffalo] because as the lake levels go up and down, you’re stressing the Earth’s crust, putting weight on one side or the other. That’s a source of stress that could give you these pretty small quakes.

SN: The images emerging from this deadly disaster are devastating.

Hough: It’s hard to watch. And it hammers home the importance of building codes. One of the problems that any place is up against is that building codes improve over time, and you’ve always got the problem of older structures. It’s really expensive to retrofit. I expect that earthquake engineers will be looking at the damage, and it will illuminate where the vulnerabilities are [in the area]. The hope is that with proper engineering, we can make the built environment safe.

Pfizer is racing to get approval for its COVID-19 vaccine, applying for emergency use authorization from the U.S. Food and Drug Administration on November 20. But the pharmaceutical giant faces a huge challenge in distributing its vaccine, which has to be kept an ultrafrosty –70° Celsius, requiring special storage freezers and shipping containers.

It “has some unique storage requirements,” says Kurt Seetoo, the immunization program manager at the Maryland Department of Public Health in Baltimore. “We don’t normally store vaccines at that temperature, so that definitely is a challenge.”

That means that even though the vaccine developed by Pfizer and its German partner BioNTech is likely to be the first vaccine to reach the finish line in the United States, its adoption may ultimately be limited. The FDA’s committee overseeing vaccines will meet on December 10 to discuss the emergency use request. That meeting will be streamed live on the agency’s web site and YouTube, Facebook and Twitter channels.

The companies are also seeking authorization to distribute the vaccine in Australia, Canada, Europe, Japan, the United Kingdom
A similar vaccine developed by Moderna and the U.S. National Institute of Allergy and Infectious Diseases also requires freezing. But it survives at a balmier –20° C, so can be kept in a standard freezer, and can even be stored at refrigerator temperatures for up to a month. Most vaccines don’t require freezing at all, but both Pfizer and Moderna’s vaccines are a new type of vaccine for which the low temperatures are necessary to keep the vaccines from breaking down and becoming useless.

Both vaccines are based on messenger RNA, or mRNA, which carries instructions for building copies of the coronavirus’ spike protein. Human cells read those instructions and produce copies of the protein, which, in turn prime the immune system to attack the coronavirus should it come calling.

So why does Pfizer’s vaccine need to be frozen at sub-Antarctica temperatures and Moderna’s does not?

Answering that question requires some speculation. The companies aren’t likely to reveal all the tricks and commercial secrets they used to make the vaccines, says Sanjay Mishra, a protein chemist and data scientist at Vanderbilt University Medical Center in Nashville.

But there are at least four things that may determine how fragile an mRNA vaccine is and how deeply it needs to be frozen to keep it fresh and effective. How the companies addressed those four challenges is likely the key to how cold the vaccines need to be, Mishra says.

The cold requirement conundrum starts with the difference in chemistry between RNA and its cousin, DNA.
One reason RNA is much less stable than DNA is due to an important difference in the sugars that make up the molecules’ backbones. RNA’s spine is a sugar called ribose, while DNA’s is deoxyribose. The difference: DNA is missing an oxygen molecule. As a result, “DNA can survive for generations,” Mishra says, but RNA is much more transient. “And for biology, that’s a good thing.”

When cells have a job to do, they usually need to call proteins into service. But like most manufacturers, cells don’t have a stockpile of proteins. They have to make new batches each time. The recipe for making proteins is stored in DNA.

Rather than risk damaging DNA recipes by putting them on the molecular kitchen counter while cooking up a batch of proteins, cells instead make RNA copies of the recipe. Those copies are read by cellular machinery and used to produce proteins.
Like a Mission Impossible message that self-destructs once it has been played, many RNAs are quickly degraded once read. Quickly disposing of RNA is one way to control how much of a particular protein is made. There are a host of enzymes dedicated to RNA’s destruction floating around inside cells and nearly everywhere else. Sticking RNA-based vaccines in the blast freezer prevents such enzymes from tearing apart the RNA and rendering the vaccine inert.

Another way the molecules’ stability differs lies in their architecture. DNA’s dual strands twine into a graceful double helix. But RNA goes it alone in a single strand that pairs with itself in some spots, creating fantastical shapes reminiscent of lollipops, hair pins and traffic circles. Those “secondary structures” can make some RNAs more fragile than others.

Yet another place that DNA’s and RNA’s chemical differences make things hard on RNA is the part of the molecules that spell out the instructions and ingredients of the recipe. The information-carry subunits of the molecules are known as nucleotides. DNA’s nucleotides are often represented by the letters A, T, C and G for adenine, thymine, cytosine and guanine. RNA uses the same A, C and G, but in place of thymine it has a different letter: uracil, or U.

“Uracil is a problem because it juts out,” Mishra says. Those jutting Us are like a flag waving to special immune system proteins called Toll-like receptors. Those proteins help detect RNAs from viruses, such as SARS-CoV-2, the coronavirus that causes COVID-19, and slate the invaders for destruction.

All these ways mRNA can fall apart or get waylaid by the immune system create an obstacle course for vaccine makers. The companies need to ensure that the RNA stays intact long enough to get into cells and bake up batches of spike protein. Both Moderna and Pfizer probably tinkered with the RNA’s chemistry to make a vaccine that could get the job done: Both have reported that their vaccines are about 95 percent effective at preventing illness in clinical trials (SN: 11/16/20; SN: 11/18/20).

While the details of each company’s approach aren’t known, they both probably fiddled slightly with the chemical letters of the mRNAs in order to make it easier for human cellular machinery to read the instructions. The companies also need to add additional RNA — a cap and tail — flanking the spike protein instructions to make the molecule stable and readable in human cells. That tampering may have disrupted or created secondary structures that could affect the RNA’s stability, Mishra says.
The uracil problem can be dealt with by adding a modified version of the nucleotide, which Toll-like receptors overlook, sparing the RNA from an initial immune system attack so that the vaccine has a better chance of making the protein that will build immune defenses against the virus. Exactly which modified version of uracil the companies may have introduced into the vaccine could also affect RNA stability, and thus the temperature at which each vaccine needs to be stored.

Finally, by itself, an RNA molecule is beneath a cell’s notice because it’s just too small, Mishra says. So the companies coat the mRNA with an emulsion of lipids, creating little bubbles known as lipid nanoparticles. Those nanoparticles need to big enough that cells will grab them, bring them inside and break open the particle to release the RNA.

Some types of lipids stand up to heat better than others. It’s “like regular oil versus fat. You know how lard is solid at room temperature” while oil is liquid, Mishra says. For nanoparticles, “what they’re made of makes a giant difference in how stable they will be in general to [maintain] the things inside.” The lipids the companies used could make a big difference in the vaccine’s ability to stand heat.

The need for ultracold storage might ultimately limit how many people end up getting vaccinated with Pfizer’s vaccine. “We anticipate that this Pfizer vaccine is pretty much only going to be used in this early phase,” Seetoo says.

The first wave of immunizations is expected to go to health care workers and other essential employees, such as firefighters and police, and to people who are at high risk of becoming severely ill or dying of COVID-19 should they contract it such as elderly people living in nursing facilities.

Pfizer has told health officials that the vaccine can be stored in special shipping containers that are recharged with dry ice for 15 days and stay refrigerated for another five days after thawing, Seetoo says. That gives health officials 20 days to get the vaccine into people’s arms once it’s delivered. But Moderna’s vaccine and a host of others that are still in testing seem to last longer at warmer temperatures. If those vaccines are as effective as Pfizer’s, they may be more attractive candidates in the long run because they don’t need such extreme special handling.