Structure-guided nanobodies block SARS-CoV-2 infection

Monoclonal antibodies are valuable weapons in the battle against COVID-19 as direct-acting antiviral agents (1). Central to virus replication cycle, the SARS-CoV-2 spike protein binds the host cell receptor and engages in virus-host membrane fusion (2). Conformational flexibility of the spike protein allows each of its receptor binding domains (RBDs) to exist in two major configurations: a “down” conformation that is thought to be less accessible to binding of many neutralizing antibodies and an “up” conformation that binds both the receptor and neutralizing antibodies (3-5). Some neutralizing antibodies bind to the RBD in the “up” conformation and compete with the receptor (6, 7), while some neutralizing antibodies bind and stabilize the “down” confor­mation to prevent the conforma­tional changes required for viral entry, thereby hindering infection (8, 9).

Unfortunately, antibody molecules can be more difficult to produce in large quantities and are relatively costly to produce. Single domain antibodies, also known as nanobod­ies, offer an opportunity to rapidly produce antiviral agents for immun­ization and for therapy. Nanobodies are easier to produce, have high thermal stability and have the potential to be administered by inha­lation.

Read more on the SLAC website

Image: Bivalent nanobodies inducing post-fusion conformation of the SARS-CoV-2 spike protein: SARS-CoV-2 spike proteins are in a fusion inactive configuration when the RBDs are in the down conformation (left). Binding of bivalent nanobody (red and green ribbons joined by yellow tether) stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the post-fusion conformation, which irreversibly inactivates the spike protein (right).

Scientists glimpse signs of a puzzling state of matter in a superconductor

Known as “pair-density waves,” it may be key to understanding how superconductivity can exist at relatively high temperatures.

Unconventional superconductors contain a number of exotic phases of matter that are thought to play a role, for better or worse, in their ability to conduct electricity with 100% efficiency at much higher temperatures than scientists had thought possible – although still far short of the temperatures that would allow their wide deployment in perfectly efficient power lines, maglev trains and so on.

Now scientists at the Department of Energy’s SLAC National Accelerator Laboratory have glimpsed the signature of one of those phases, known as pair-density waves or PDW, and confirmed that it’s intertwined with another phase known as charge density wave (CDW) stripes – wavelike patterns of higher and lower electron density in the material.

Observing and understanding PDW and its correlations with other phases may be essential for understanding how superconductivity emerges in these materials, allowing electrons to pair up and travel with no resistance, said Jun-Sik Lee, a SLAC staff scientist who led the research at the lab’s Stanford Synchrotron Radiation Lightsource (SSRL).

Read more on the SLAC website

Image: SLAC scientists used an improved X-ray technique to explore exotic states of matter in an unconventional superconductor that conducts electricity with 100% efficiency at relatively high temperatures. They glimpsed the signature of a state known as pair density waves (PDW), and confirmed that it intertwines with another phase known as charge density wave (CDW) stripes – wavelike patterns of higher and lower electron density in the material. CDWs, in turn, are created when spin density waves (SDWs) emerge and intertwine.

Credit: Jun-Sik Lee/SLAC National Accelerator Laboratory

Researchers search for clues to COVID-19 treatment

Two groups of researchers drew on SLAC tools to better understand how to target a key part of the virus that causes COVID-19

Vaccination, masks and physical distancing help limit the spread of COVID-19 – but, researchers say, the disease is still going to infect people, and doctors are still going to need better medicines to treat patients. This may be especially true for cancer patients and other at-risk people who may lack a sufficiently strong immune system to benefit from the vaccine. 

Now, two teams working in part at the Department of Energy’s SLAC National Accelerator Laboratory have found some clues that could, down the road, lead to new COVID drugs. 

The researchers, from John Tainer’s lab at MD Anderson Cancer Center and James Fraser’s group at the University of California, San Francisco, focused on a molecular structure that is common to all coronaviruses but has proven especially troublesome in the case of the virus that causes COVID-19. The structure contributes both to the virus’s ability to replicate and to immune system overreactions that have proven particularly deadly.

The trouble, Fraser said, is that scientists don’t know what kinds of molecules would bind to the structure, known as the Nsp3 macrodomain, let alone how to combine such molecules to interfere with its deadly work. 

To remedy that problem, Fraser’s group screened several thousand molecules at facilities including SLAC’s Stanford Synchrotron Radiation Lightsource (SSRL) to see where and how well the molecules bound to crystallized forms of Nsp3. The team combined those results with computer models to understand how the molecules might affect the structure of the macrodomain and whether they might help inhibit its function. 

Read more on the SLAC website

Blood disorder mechanism discovered

G6PD deficiency affects about 400M people worldwide and can pose serious health risks. Uncovering the causes of the most severe cases could finally lead to treatments.

With a name like glucose-6-phosphate dehydrogenase deficiency, one would think it is a rare and obscure medical condition, but that’s far from the truth. Roughly 400 million people worldwide live with potential of blood disorders due to the enzyme deficiency. While some people are asymptomatic, others suffer from jaundice, ruptured red blood cells and, in the worst cases, kidney failure. 

Now, a team led by researchers at the Department of Energy’s SLAC National Accelerator Laboratory has uncovered the elusive mechanism behind the most severe cases of the disease: a broken chain of amino acids that warps the shape of the condition’s namesake protein, G6PD. The team, led by SLAC Professor Soichi Wakatsuki, report their findings January 18th in Proceedings of the National Academy of Sciences

Read more on the SLAC website

Image: The G6PD enzyme plays a crucial role in red blood cells, removing molecules such as hydrogen peroxide from the body. In some cases, mutations can bend the molecule awkwardly, interfering with G6PD’s function. In the worst cases, the mutations lead red blood cells to rupture.

Credit: Mio Wakatsuki, from protein images by Naoki Horikoshi/SLAC National Accelerator Laboratory

Scientists probe the chemistry of a single battery electrode particle both inside and out

The results show how a particle’s surface and interior influence each other, an important thing to know when developing more robust batteries.

The particles that make up lithium-ion battery electrodes are microscopic but mighty: They determine how much charge the battery can store, how fast it charges and discharges and how it holds up over time – all crucial for high performance in an electric vehicle or electronic device.

Cracks and chemical reactions on a particle’s surface can degrade performance, and the whole particle’s ability to absorb and release lithium ions also changes over time. Scientists have studied both, but until now they had never looked at both the surface and the interior of an individual particle to see how what happens in one affects the other.

Read more on the SSRL (SLAC National Accelerator Laboratory) website

Image: Images made with an X-ray microscope show particles within a nickel-rich layered oxide battery electrode (left). In a SLAC study, scientists welded a single charged particle to the tip of a tungsten needle (right) so they could probe its surface and interior with two X-ray instruments. The particle is about the size of a red blood cell. (S. Li et al., Nature Communications, 2020)

Cross-β Structure – a Core Building Block for Streptococcus mutans Functional Amyloids

Most amyloids1 are misfolded proteins, having enormous variety in native structures. Pathological amyloids are implicated in diseases including Alzheimer’s disease and many others.  They are characterized by long, unbranched fibrillar structure, enhanced birefringence on binding Congo red dye, and cross-β structure – β-strands running approximately perpendicular to the fibril axis, forming long β-sheets running in the direction of the axis.  Fiber diffraction patterns from amyloids are marked by strong intensity at about 4.8 Å in the meridional direction (parallel to the fibril axis), corresponding to the separation of strands in a β-sheet, and in many cases broader but distinct equatorial intensity at about 10 Å.  The 10 Å intensity (whose position may vary considerably) comes from the distance between stacked β-sheets.  This stacking is characteristic of the many amyloids formed by small peptides, including peptide fragments of larger amyloidogenic proteins.  While some authors have required the 10 Å intensity to characterize an amyloid, it is not strictly necessary, since architecturally more complex examples have been found of Congo-red-staining fibrils with cross-β structure, but without the stacked-sheet structure, and consequently without the 10 Å intensity on the equator.

Amyloids do not always stem from protein misfolding.  Organisms across all kingdoms utilize functional amyloids in numerous biological processes.  Bacteria are no exception. Bacterial amyloids contribute to biofilm formation and stability.  Tooth decay is the most common infectious disease in the world.  A major etiologic agent, Streptococcus mutans, is a quintessential biofilm dweller that produces at least three different amyloid-forming proteins, adhesins P1 and WapP, and the cell density and competence regulator Smu_63c2.  The naturally occurring truncation derivatives of P1 and WapA, C123 and AgA, represent the amyloidogenic moieties, and a new paradigm of Gram-positive bacterial adhesins is emerging of adhesins having dual functions in monomeric and amyloid forms. While each S. mutans protein possesses considerable β-sheet structure, the tertiary structures of each protein are quite different (Fig. 1).  This study further characterized S. mutans amyloids and addressed the ongoing debate regarding the underlying structure and assembly of bacterial amyloids including speculation that they are structurally dissimilar from better-characterized amyloids.

Read more on the SSRL website

Image: Crystal or predicted 3D structures of S. mutans C123 (left), AgA (center), and Smu_63c (right).

Structure and functional binding epitopes of VISTA

V-domain Ig Suppressor of T-cell Activation (VISTA) is an immune checkpoint protein involved in the regulation of T cell activity. Checkpoint proteins are overexpressed by cancer cells or surrounding immune cells and prevent anti-tumor activity by co-opting natural regulation mechanisms to escape immune clearance. Compared to healthy tissues, VISTA is upregulated on tumor infiltrating leukocytes, including high expression on myeloid-derived suppressor cells (MDSCs). Through VISTA signaling, these inhibitory immune cells prevent effective antigen presentation and indirectly promote tumor growth. VISTA is implicated in a number of human cancers including skin (melanoma), prostate, colon, pancreatic, ovarian, endome­trial, and non-small cell lung. VISTA is a known member of the B7 protein family but the mechanism of action is still unclear as VISTA has been shown to function as both a ligand1,2 and a receptor3.  In the model of VISTA as a receptor, the proposed ligand of interaction is V-set and immunoglobulin domain containing 3 (VSIG3)4,5.

>Read more on the SSRL website

Image: Structure of human VISTA with extended C-C’ loop (blue), mapped VSTB/VSIG3 binding epitope (red), and disulfide bonds (yellow).

The role of ‘charge stripes’ in superconducting materials

The studies could lead to a new understanding of how high-temperature superconductors operate.

High-temperature superconductors, which carry electricity with zero resistance at much higher temperatures than conventional superconducting materials, have generated a lot of excitement since their discovery more than 30 years ago because of their potential for revolutionizing technologies such as maglev trains and long-distance power lines. But scientists still don’t understand how they work.
One piece of the puzzle is the fact that charge density waves – static stripes of higher and lower electron density running through a material – have been found in one of the major families of high-temperature superconductors, the copper-based cuprates. But do these charge stripes enhance superconductivity, suppress it or play some other role?
In independent studies, two research teams report important advances in understanding how charge stripes might interact with superconductivity. Both studies were carried out with X-rays at the Department of Energy’s SLAC National Accelerator Laboratory.

>Read more on the LCLS at SLAC website

Image: This cutaway view shows stripes of higher and lower electron density – “charge stripes” – within a copper-based superconducting material. Experiments with SLAC’s X-ray laser directly observed how those stripes fluctuate when hit with a pulse of light, a step toward understanding how they interact with high-temperature superconductivity.
Credit: Greg Stewart/SLAC National Accelerator Laboratory

Winning the fight against influenza

Annual influenza epidemics and episodic pandemics continue to cause widespread illness and mortality. The World Health Organization estimates that annual influenza epidemics cause around 3–5 million cases of severe illness and up to 650,000 deaths worldwide. Seasonal influenza vaccination still remains the best strategy to prevent infection, but the vaccines that are available now offer a very limited breadth of protection. Human broadly neutralizing antibodies (bnAbs) that bind to the hemagglutinin (HA) stem region provide hope for a universal vaccine (Figure 1a)1,2. Binding of these bnAbs prevents the pH-induced conformational changes that are required for viral fusion in the endosomal compartments of target cells in the respiratory tract and, hence, viral entry in our cells.

>Read more on the SSRL at SLAC website

Image: Complex of Influenza virus HA with (a) Fab CR6261, (b) llama single domain antibody SD36, and (c) JNJ4796.

In a first, researchers identify reddish coloring in an ancient fossil mouse

X-rays reveal an extinct mouse was dressed in brown to reddish fur on its back and sides and had a tiny white tummy.

Researchers have for the first time detected chemical traces of red pigment in an ancient fossil – an exceptionally well-preserved mouse, not unlike today’s field mice, that roamed the fields of what is now the German village of Willershausen around 3 million years ago.
The study revealed that the extinct creature, affectionately nicknamed “mighty mouse” by the authors, was dressed in brown to reddish fur on its back and sides and had a tiny white tummy. The results were published today inNature Communications.
The international collaboration, led by researchers at the University of Manchester in the U.K., used X-ray spectroscopy and multiple imaging techniques to detect the delicate chemical signature of pigments in this long-extinct mouse.

>Read more on the SSRL at SLAC Lab website

Image: In this image showing the fossil chemistry of an ancient mouse, blue represents calcium in the bones, green is the element zinc which has been shown to be important in the biochemistry of red pigment and red is a particular type of organic sulfur. This type of sulfur is enriched in red pigment. When combined, regions rich in both zinc and sulfur appear yellow on this image, showing that the fur on this animal was rich in the chemical compounds that are most probably derived from the original red pigments produced by the mouse. (10.1038/s41467-019-10087-2)

Doubling the DNA alphabet

Implications for life in the universe and DNA storage

Life on Earth is dictated by the DNA alphabet comprised of only four DNA bases or letters: A, T, G and C. It has long been of interest to understand whether there is something very special about the four letters that comprise DNA and whether this is the only code that could support life. At a basic level, this question can be addressed by examining an expanded alphabet and determining the properties of DNA including additional synthetic letters. This study impacts our current understanding of terrestrial DNA and suggests that extraterrestrial life forms could have evolved using a different genetic code than found here on Earth. The work has immediate applications in synthetic biology for the creation of new molecules and greatly expands the ability to store information in DNA.

Now, in breakthrough work, funded by NASA, NSF and NIGMS, Dr. Steven Benner at the Foundation for Applied Molecular Evolution, in collaboration with Dr. Millie Georgiadis at the Indiana University School of Medicine, and colleagues at biotechnology companies and other universities, have provided evidence that the standard DNA code can be expanded to include eight letters forming “hachimoji DNA” (“hachi” eight and “moji” letter in Japanese) using four novel synthetic nucleobases (B, S, P and Z) in addition to A, T, C and G and still retain critical features of natural DNA1,2. Structurally, hachimoji DNA can adopt a standard double helical form of DNA and retain Watson-Crick complementary base pairing, which allows the expanded DNA to be faithfully replicated and transcribed by polymerases to produce hachimoji DNA copies and hachimoji RNA. These properties are essential for a genetic system that can support life.

>Read more on the Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC website

Image: Crystal structure of a double helix built from eight hachimoji building blocks, G (green), A (red), C (dark blue), T (yellow), B (cyan), S (pink), P (purple), and Z (orange). The first four building blocks are found in human DNA; the last four are synthetic, and possibly present in alien life. Each strand of the double helix has the sequence CTTAPCBTASGZTAAG. Notable is the geometric regularity of the pairs, a regularity that is needed for evolution.

Cause of cathode degradation identified for nickel-rich materials

Combination of research methods reveals causes of capacity fading, giving scientists better insight to design advanced batteries for electric vehicles

A team of scientists including researchers at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory and SLAC National Accelerator Laboratory have identified the causes of degradation in a cathode material for lithium-ion batteries, as well as possible remedies. Their findings, published on Mar. 7 in Advanced Functional Materials, could lead to the development of more affordable and better performing batteries for electric vehicles.

Searching for high-performance cathode materials
For electric vehicles to deliver the same reliability as gas vehicles they need lightweight yet powerful batteries. Lithium-ion batteries are the most common type of battery found in electric vehicles today, but their high cost and limited lifetimes are limitations to the widespread deployment of electric vehicles. To overcome these challenges, scientists at many of DOE’s national labs are researching ways to improve the traditional lithium-ion battery.

>Read more on the NSLS-II at Brookhaven Lab website

Image: Members of the Brookhaven team are shown at NSLS-II’s ISS beamline, where part of the research was conducted. Pictured from front to back are Eli Stavitski, Xiao-Qing Yang, Xuelong Wang, and Enyuan Hu.

Mechanism of thiopurine resistance in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is an aggressive lymphoid malignancy that is currently the leading cause of cancer in pediatric patients1. Despite intensified chemotherapy regimens, the cure rates of ALL only approaches 40%2. Specific mutations in the cytosolic 5’-nucleotidase II (NT5C2) gene are present in about 20% of relapsed pediatric T-ALL and 3-10% of relapsed B-precursor ALL cases3,4.

NT5C2 is a cytosolic nucleotidase that maintains intracellular nucleotide pool levels by exporting excess purine nucleotides out of the cell5.  NT5C2 can also dephosphorylate and inactivate the metabolites of the 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP) commonly used to treat ALL6. Thus, relapse associated activating mutations in NT5C2 confer resistance to 6-MP and 6-TG chemotherapy. Upon allosteric activation, a disordered region of NT5C2 adopts a helical configuration (helix A) and facilitates substrate binding and catalysis (Fig. 1a)7.  Mutations in this regulatory region of NT5C2 have been modeled to strongly activate NT5C2.  However, the majority of NT5C2 mutations associated with relapsed ALL do not occur in this region.
To better understand the mechanisms by which these gain-of function NT5C2 mutations lead to increased nucleotidase activity, Dieck, Tzoneva, Forouhar and colleagues investigated additional regulatory elements that may control NT5C2 activation.  They collected crystallographic data for several mutant NT5C2 homotetramers at SSRL (NT5C2-537X D52N/D407A in active state (BL9-2), NT5C2-Q523X D52N in basal state and in active state (BL14-1) and full-length NT5C2 R39Q/D52N in basal state (BL12-2)) and used the structural information as a guide in the understanding of the mechanistic details.

>Read more on the Stanford Synchrotron Radiation Lightsource website

Figure (a) A ribbon diagram of the active structure of NT5C2 WT, in which the allosteric helix A (αA) is shown in dark purple. The N and C termini amino acids (S4 and S488), and the termini amino acids (L402 and R421) of the disordered region in the arm segment are also labeled. Panels b and c shows ribbon and surface (for subunit B) depictions of basal (b) and active dimers (c) of WT.

Structural basis of neurosteroid anesthetic action on GABAA receptors

Type A γ-aminobutyric acid receptors (GABAARs) control neuronal excitability1. They are targets for the treatment of neurological diseases and disorders and also for general anesthetics. The underlying mechanisms of these drugs’ action on GABAARs remain to be determined.
One of the mechanisms is to potentiate function of GABAARs via binding to the transmembrane domain (TMD)2. Ample experimental evidence suggests that the TMD of GABAARs harbors sites for the primary actions of general anesthetics and neurosteroids. The TMD plays an essential role in functional transitions among the resting, activated, and desensitized states of these Cl-conducting channels.
Alphaxalone (5α-pregnan-3α-ol-11,20 dione) is a potent neurosteroid anesthetic. The anxiolytic, anticonvulsant, analgesic, and sedative-hypnotic effects of alphaxalone have been linked to its potentiation of GABA-evoked currents and direct activation of GABAARs3. However, the data about the alphaxalone binding site in GABAARs and the underlying structural basis of alphaxalone’s action are sparse.

>Read more on the Stanford Synchrotron Radiation Lightsource at SLAC

Figure: Alphaxalone-induced structural changes at the bottom of the TMD (a) Bottom view of overlaid TM1-TM2 structures of the apo (orange) and alphaxalone-bound (cyan) α1GABAAR chimera. (b) Side view of overlaid structures of apo (principal subunit – gold; complementary subunit – orange) and alphaxalone-bound (principal subunit – blue; complementary subunit – cyan) α1GABAAR chimera. For clarity, only TM2 and TM3 are shown in the principal subunit and only TM1 and TM2 are shown in the complementary subunit. The arrow highlights structural perturbations originating from the alphaxalone binding site near W246 through the TM1-TM2 linker to the pore-lining residues P253 (-2′) and V257 (2′). (c) The 2FO-FC electron density maps (blue mesh, contoured at 1 σ) covering TM1-TM2 in the apo (left) and alphaxalone-bound (right) α1GABAAR chimera. The sidechains are shown only for residues W246 to V257 (2′).

Untangling a strange phenomenon in lithium-ion batteries

New research offers the first complete picture of why a promising approach of stuffing more lithium into battery cathodes leads to their failure.

A better understanding of this could be the key to smaller phone batteries and electric cars that drive farther between charges.
The lithium-ion batteries that power electric vehicles and phones charge and discharge by ferrying lithium ions back and forth between two electrodes, an anode and a cathode. The more lithium ions the electrodes are able to absorb and release, the more energy the battery can store.
One issue plaguing today’s commercial battery materials is that they are only able to release about half of the lithium ions they contain. A promising solution is to cram cathodes with extra lithium ions, allowing them to store more energy in the same amount of space. But for some reason, every new charge and discharge cycle slowly strips these lithium-rich cathodes of their voltage and capacity.
A new study provides a comprehensive model of this process, identifying what gives rise to it and how it ultimately leads to the battery’s downfall. Led by researchers from Stanford University and the Department of Energy’s SLAC National Accelerator Laboratory and Lawrence Berkeley National Laboratory, it was published today in Nature Materials.

>Read more on the Stanford Synchrotron Radiation Lightsource (SSRL)

Image: A mysterious process called oxygen oxidation strips electrons from oxygen atoms in lithium-rich battery cathodes and degrades their performance, shown at left. Better understanding this property and controlling its effects could lead to better performing electric vehicles.
Credit: Gregory Stewart/SLAC National Accelerator Laboratory)

Copper mobilization and immobilization

… along an organic matter and redox gradient- insights from a mofette site.

The metal copper (Cu) is known to be an essential trace element for many organisms but it is also considered a severe contaminant at higher concentrations. Especially in soils with changing redox conditions, Cu binding mechanisms and, thus, Cu mobility are hard to predict. The metal is known to have a high affinity to soil organic matter (SOM), i.e., it can either be sequestered by adsorption to solid-phase organic matter or mobilized by complexation with dissolved organic matter. Under reducing conditions, Cu(II) can also be reduced to Cu(I) via biotic and abiotic processes and precipitate in the form of sulfidic minerals.
>Read more on the SSRL website
Image: Picture of the investigated mofette site (left) and Cu sorption isotherms determined for mofette, transitions, and reference soil in a Cu spike experiment (right).
Credit: Reprinted with permission from Mehlhorn et al. 2018, ES&T, DOI: 10.1021/acs.est.8b02668, Copyright 2018 American Chemical Society