Tag: X-ray crystallography

Synchrotron light reveals how a plant enzyme reshapes sugars to drive essential biological reactions

Synchrotron light reveals how a plant enzyme reshapes sugars to drive essential biological reactions

Using XALOC beamline at ALBA, researchers from the Institute of Biocomputation and Physics of Complex Systems (BIFI), at the University of Zaragoza, have discovered an unexpected way in which a plant enzyme activates sugars during a fundamental biochemical reaction. The findings, published in Nature Communications, challenge long-standing assumptions about how glycosyltransferase enzymes work and provide new foundations for biotechnological innovation.

Sugar-modifying enzymes play a central role in life. They control how sugars are attached to proteins and other molecules, a process that influences cell communication, development, immunity and responses to stress. In plants, these reactions are essential for building cell walls and regulating growth, while in humans similar enzymes are linked to disease processes and the effectiveness of therapeutic antibodies. Understanding exactly how these enzymes work at the molecular level is crucial not only for basic biology, but also for improving biomedicine, agriculture and industrial biotechnology.

The study of this group from BIFI at the University of Zaragoza focuses on FUT11, a fucosyltransferase enzyme from the model plant Arabidopsis thaliana. Glycosyltransferases such as FUT11 catalyse the formation of glycosidic bonds by transferring a sugar from a donor molecule to an acceptor. Traditionally, scientists assumed that during this reaction the acceptor sugar remained largely passive, maintaining a stable shape while the enzyme activated the donor. Using high-resolution structural data collected at the XALOC beamline – one of the ALBA’s instruments for X-ray crystallography-, the researchers were able to visualise FUT11 bound to its substrates and discovered a very different picture.

The crystal structures collected at ALBA (BL13 XALOC) provided the structural framework for mechanistic interpretation, and the accompanying atomistic simulations indicated that FUT11 actively promotes a transient distortion (puckering) of the acceptor sugar ring away from its most stable chair conformation. In these simulations, the catalytic base—Glu158—acts not only as the proton abstractor but also as a conformational effector: its interactions bias the innermost GlcNAc into a reactive, puckered state that better aligns the acceptor hydroxyl for nucleophilic attack and efficient bond formation.

Read more on the ALBA website

Image: Researchers Víctor Taleb, María Bort, Ramón Hurtado from BIFI

Credit: Unizar

ALS Captures Structure of Engineered Protein, Opening New Options to Treat IBD

ALS Captures Structure of Engineered Protein, Opening New Options to Treat IBD

According to the Centers for Disease Control and Prevention, Inflammatory Bowel Disease (IBD) affects more than three million people across the United States, costing the nation’s healthcare system about $8.5 billion in 2018. IBD occurs when the body’s immune system mistakenly attacks healthy bowel tissue, leading to inflammation and damage.

The exact cause of IBD is unknown but is characterized by long-term inflammation. While many factors can influence inflammation, Interleukin-10 (IL-10), a specialized protein, plays a role in regulating the immune response in the human body and has potential to treat inflammation-related conditions. Unfortunately, wild-type IL-10 is complicated. It possesses a dual nature and can initiate both pro- and anti-inflammatory pathways. It is also ephemeral, only lasting three hours in the human body.

“Natural proteins can be problematic as biotherapeutics, because these compounds often have a limited half-life and toxicity,” said Glen Spraggon, executive director of Structure Bioinformatics and Data Science at the Novartis Biologics Research Center and senior author on the study. “We decided to try to combine the positive properties of antibodies—half-life and good manufacturability—with the functional properties of IL-10.”

Spraggon and his colleagues grafted a modified IL-10 protein into the complementary determining regions (CDR) of an antibody. Through this process, they engineered six graft variants. One graft (GFT-IL10M) had the desired properties, but the actual molecule structure of the design remained unclear. The joints in the graft connecting the antibody and IL-10 are incredibly flexible. The team crystallized the engineered sample and analyzed it at the Advanced Light Source Beamline 5.0.3. They used the diffraction data to define the three-dimensional structure of the IL-10 fusion protein at the atomic level.

“We love to have a visual to understand the molecular structure of what we have actually created in the lab,” said Spraggon. “This structure confirmed how, with this fusion approach, we largely change the signaling profile of the molecule, biasing it away from its pro-inflammatory nature towards anti-inflammatory.”

Read more on ALS website

Image: Molecular rendering of the engineered graft of IL-10 (red) with an antibody (blue/yellow). Structural analysis of GFT-IL10M using X-ray Crystallography at the ALS provided insight into its improved anti-inflammatory properties. The designed biotherapeutic enhanced monocyte activation whilst minimizing pro-inflammatory signaling observed in wild-type IL-10which also stimulates B, T, and NK white-blood cells.

Credit: Michael DiDonato/Novartis Biomedical Research

Closing a gap in the race toward HIV vaccine development

Closing a gap in the race toward HIV vaccine development

Work to develop a vaccine to protect against human immunodeficiency virus (HIV) has been underway for four decades but we still have no effective vaccine. Treatments have been developed that allow people infected with HIV to live long and healthy lives, but a vaccine could prevent new infections, affecting about 1.3 million people annually worldwide, and could potentially eradicate the disease.

Unfortunately, vaccine development has been plagued by challenges related to the evasive tactics of the virus, which mutates very quickly, and difficulties in obtaining a structure for the main envelope protein of the virus, Env, which was finally solved by electron microscopy and crystallography in 2013. HIV vaccine researchers have come to the conclusion that ideal vaccine immunogens designed to generate a protective immune response should elicit antibodies that can recognize HIV via a number of target sites on the Env protein. Broadly neutralizing antibodies to each of these sites have been identified in some people infected with HIV-1 and in some animal models but efforts to elicit this broad antibody recognition of the diverse HIV strains and subtypes in response to a vaccine have not been successful.

A recent study from a group at the Ragon Institute, Scripps Research Institute, Leipzig University, La Jolla Institute for Immunology, UC San Diego, Moderna, and Massachusetts Institute of Technology provides insights into antibody-based HIV vaccine development that could lead to the identification of vaccine immunogen candidates to elicit such broadly neutralizing antibodies.

Scripps Research investigators used resources of the National Institute of General Medical Science and National Cancer Institute Structural Biology Facility (GM/CA) at beamline 23-ID-B of the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.

The research started with the observation that a broadly neutralizing antibody called 10E8 has excellent HIV neutralization properties but does not recognize self-antigens. This is an undesirable feature, as earlier antibodies to this membrane-proximal external region (MPER) site did. During an immune response that leads to the development of antibodies, precursor B cells that make a certain type of antibody are activated by the antigen they recognize (e.g., a viral protein), undergo mutations that increase their affinity for the antigen, and then start expanding to produce more of these cells.

With 10E8, the researchers identified precursors of the antibody, before these affinity mutations, and engineered immunogens to specifically activate these rare 10E8-class precursor B cells. This technique is called germline targeting.

To test their new immunogens, the researchers created mice that express the 10E8-class precursor B cells, which are normally only found in humans. They found that these B cells were functioning normally in mice, but when they immunized the mice with an immunogen designed to activate their targeted 10E8-class precursors B cells, they didn’t see the expansion they had hoped for, even though they could show that the B cells had high enough affinity to be activated.

Read more on APS website

Image: Orientation of mature 10E8 Fab in relation to the MPER peptide (heavy chain, white; light chain, dark gray; MPER peptide, purple; top left) and surface rendering and positioning of the critical YxFW residues of HCDR3 (bottom left), representative GT10.2-10E8UCAH mAb in complex with a glycan-knockout (KO) version of 10E8-GT10.2 (heavy chain, yellow; light chain, dark gray; targeted MPER graft with GT mutations, purple; top center; structure aligned and oriented to the MPER peptide as in the top left) and designed binding pocket (green) and engagement of the GT10.2-10E8-UCAH mAb HCDR3 (yellow) compared to mature 10E8 HCDR3 (white; bottom center) and representative GT10.2-WT mAb in complex with 10E8-GT10.2 (heavy chain, red; light chain, dark gray; targeted MPER graft with GT mutations, purple; top right) and binding pocket (green; bottom right).

Taking candid shots of radical proteins

Taking candid shots of radical proteins

Scientists capture how radical electrons influence protein structure before they have time to react

Some enzymes in the body carry radicals, chemical groups with highly reactive unpaired electrons, to catalyse biochemical reactions, but it has proven challenging to study the structure of these enzymes with the radicals intact. The X-ray crystallography techniques conventionally used to study protein structure introduce ‘X-ray damage’ that would neutralise radicals and alter the protein structure. To study how radicals influence proteins, researchers turned to a ribonucleotide reductase enzyme subunit called R2 that uses a radical to synthesise DNA bases. The team previously used X-ray crystallography at beamline I24 and small angle X-ray scattering at beamline B21 of the Diamond Light Source to solve the structure of this enzyme without safeguarding the radical. In the recent study, they harnessed X-ray free electron laser (XFEL) serial femtosecond crystallography at the Linac Coherent Light Source in collaboration with Diamond’s XFEL Hub to zero in on the radical. With XFEL, they used X-rays to rapidly capture the structure of the protein within femtoseconds — 1015 times quicker than a second and too quick for X-rays to neutralise the radical or distort protein structure. By comparing the enzyme with and without the radical, they revealed that the presence of the unpaired electron greatly influences the structure of the enzyme’s active site. Their research will allow them to explore the workings of this and related enzymes in finer detail and holds promise for designing drugs that target radical enzymes in cancer cells and infectious microbes.

Read more on the Diamond website

Image credit: Martin Högbom

Picking up good vibrations – of proteins – at CHESS

Picking up good vibrations – of proteins – at CHESS

A new method for analyzing protein crystals – developed by Cornell researchers and given a funky two-part name – could open up applications for new drug discovery and other areas of biotechnology and biochemistry.

The development, outlined in a paper published March 3 in Nature Communications, provides researchers with the tools to interpret the once-discarded data from X-ray crystallography experiments – an essential method used to study the structures of proteins. This work, which builds on a study released in 2020, could lead to a better understanding of a protein’s movement, structure and overall function.

Protein crystallography produces bright spots, known as Bragg peaks, from the crystals, providing high-resolution information about the shape and structure of a protein. This process also captures blurry images – patterns and clouds related to the movement and vibrations of the proteins – hidden in the background of the Bragg peaks.

These background images are typically discarded, with priority given to the bright Bragg peak imagery that is more easily analyzed.

“We know that this pattern is related to the motion of the atoms of the protein, but we haven’t been able to use that information,” said lead author Steve Meisburger, Ph.D. ’14, a former postdoctoral researcher in the lab of Nozomi Ando, M.S. ’04, Ph.D. ’09, associate professor of chemistry and chemical biology in the College of Arts and Sciences. “The information is there, but we didn’t know how to use it.  Now we do.”

Meisburger worked closely with Ando to develop the robust workflow to decode the weak background signals from crystallography experiments called diffuse scattering. This allows researchers to analyze the total scattering from crystals, which depends on both the protein’s structure and the subtle blur of its movements.

Their two-part method – which the team dubbed GOODVIBES and DISCOBALL – simultaneously provides a high-resolution structure of the protein and information on its correlated atomic movements.

GOODVIBES analyzes the X-ray data by separating the movements – subtle vibrations – of the protein from other proteins that might be moving around it. DISCOBALL independently validates these movements for certain proteins directly from the data, allowing researchers to trust the results from GOODVIBES and understand what the protein might be doing.

Read more on CHESS website

Image: Meisburger, Case, & Ando (2020) Nat Commun 11, 1271

Sirius helps reveal previously unknown process of maturation for key protein in SARS-CoV-2 replication

Sirius helps reveal previously unknown process of maturation for key protein in SARS-CoV-2 replication

Researchers at USP in São Carlos combined cutting-edge technologies and demonstrated that a molecule targeted by medications behaves differently than previously theorized.

A group of researchers from the University of São Paulo in São Carlos has just presented their findings from research indicating a new understanding of the maturation process and how inhibitors act upon the Mpro protein, an essential component in the life cycle of the Sars-CoV-2 virus and the target of various efforts to develop medications to treat Covid-19. Their results appear in an article entitled “An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition,” published in the journal Nature Communications (https://doi.org/10.1038/s41467-023-37035-5).

Mpro is an abbreviation for main protease, because of its importance to the virus. Today, two medications are available which interact with this molecule to treat covid-19. Still, some of the processes in this protein’s activity are not yet entirely understood, and this was the object of the study undertaken at Sirius.

As part of the role it plays in the life cycle of the Sars-CoV-2 virus, Mpro undergoes a series of modifications until it reaches its final form. Part of this process had already been described by the group from São Carlos, directed by Professor Glaucius Oliva.

André Godoy, who led the group, was one of the first external users of Sirius, the cutting- synchrotron light source planned and built by the Brazilian Center for Research in Energy and Materials (CNPEM), an organization overseen by the Ministry of Science, Technology and Innovation (MCTI).

In September 2020 he brought approximately 200 crystals containing proteins from the Sars-CoV-2 virus for analysis in the Manacá beamline, which was developed for experiments involving X-ray diffraction crystallography. “The Manacá beamline was the first research station to open at Sirius, as the result of a task-force effort at the CNPEM to support research exploring molecular mechanisms related to covid-19. This is one of the publications that resulted from this effort,” explains Harry Westfahl, Director of the Brazilian Synchrotron Light National Laboratory (LNLS).

Read more on the LNLS website

Image: Cryomicroscopy map of the Mpro dimer interacting with the N-terminal. Image obtained from analyses conducted at Diamond and Sirius by the USP São Carlos group

MAX IV research contributes to the development of new cancer drugs

MAX IV research contributes to the development of new cancer drugs

In the battle against cancer, scientists from the drug discovery company Sprint Bioscience and researchers from MAX IV have taken important steps together toward developing new and more efficient cancer drugs with the help of fragment screening by X-ray crystallography.

Cancer accounts for nearly one out of six deaths yearly. It begins when one or more genes in a cell mutate, creating an abnormal protein or preventing a protein’s formation.

Therefore, you need to start at the protein level to fight cancer.

Sprint Bioscience is working to develop new drug candidates by identifying small molecules (fragments) that can bind targeted cancer proteins. In collaboration with researchers from the FragMAX team at MAX IV during 2019 and 2020, Sprint Bioscience optimised and developed a protein crystallisation system corresponding to a cancer protein chosen by the company.

Read more on MAX IV website

Image: Crystal incubated with fragment XB00143 mounted on the BioMAX beamline during the screening campaign.

Credit: Sprint Bioscience/BioMAX

Treating COVID-19 by inhibiting viral replication

Treating COVID-19 by inhibiting viral replication

When SARS-CoV-2, the virus that causes COVID-19, enters a person’s cells, it hijacks those cells to make more viruses. First SARS-CoV-2 releases its RNA into the host cell. Then the host ribosomes translate the viral RNA into two giant protein chains (polyproteins). One protein in the giant chain, called MPro, cleaves the chain into smaller proteins, which help create more viruses and, therefore, more infection. Because of MPro’s role in initiating the viral replication process, the protein has become a target for antiviral drug developers. Recently, a team of scientists using high-brightness x-rays at the U.S. Department of Energy’s Advanced Photon Source (APS) has determined x-ray crystallographic structures of MPro cleaving the polyprotein at ten cleavage sites. Their findings, published in the journal Nature Communications, provide information about the mechanistic steps and molecular interactions that initiate viral replication, which can be used to inform antiviral therapeutic development for COVID-19, as well as other conditions for which MPro may be responsible.

Viruses can’t reproduce on their own; they need a human or animal cell to make other viruses and continue their infectious rampage. The SARS-CoV-2 virus, which causes COVID-19, employs its spike protein to enter a human cell. Once inside, the virus’s protective coating dissolves, and it dumps its genetic material—RNA—into the host cell. This RNA contains all the instructions the virus needs to replicate. What’s more, it comes in a handy form that is ready for a human cell to translate into proteins that will compose the next generation of viruses.

The SARS-CoV-2 RNA includes instructions for four proteins that make up the virus’s structure—its spike protein, protective coating, and the like—and sixteen proteins that replicate the virus. The replication process begins when the host’s ribosomes translate the replication genes into two gigantic protein chains called polyproteins.

Before replication can continue, however, these gigantic chains must be chopped up into their constituent proteins. Remarkably, the molecule that does the chopping is itself contained in the polyprotein and must hack its way out of the chain before attending to its neighbors.

Read more on the APS website

Image: Fig. 1. The amino acid residues preceding the SARS-CoV-2 polyprotein cleavage site between non-structural proteins nsp10 and nsp11 are shown in yellow. These residues are bound within the Mpro acceptor active site groove (grey semitransparent molecular surface).

#SyncroLightAt75 – Structure of the Ribosome

#SyncroLightAt75 – Structure of the Ribosome

Along with Ada Yonath and Thomas Steitz,Venkatraman Ramakrishnan from the MRC Laboratory of Molecular Biology in Cambridge, UK was awarded the 2009 Nobel Prize in Chemistry for determining the structure of the ribosome, one of the largest and most important molecules in the cell. X-ray crystallography experiments that enabled elucidation of the ribosome structure used synchrotron light from a number of light sources worldwide, each with unique capabilities, including the Swiss Light Source SLS.

Read more on the PSI website

Image: Interior view of the experimental hall at the Swiss Light Source SLS

Credit: Photo: H.R. Bramaz/PSI

DOE funds pilot study focused on biosecurity for bioenergy crops

DOE funds pilot study focused on biosecurity for bioenergy crops

Research into threats from pathogens and pests would speed short-term response and spark long-term mitigation strategies

The U.S. Department of Energy’s (DOE) Office of Science has selected Brookhaven National Laboratory to lead a new research effort focused on potential threats to crops grown for bioenergy production. Understanding how such bioenergy crops could be harmed by known or new pests or pathogens could help speed the development of rapid responses to mitigate damage and longer-term strategies for preventing such harm. The pilot project could evolve into a broader basic science capability to help ensure the development of resilient and sustainable bioenergy crops as part of a transition to a net-zero carbon economy.

The idea is modeled on the way DOE’s National Virtual Biotechnology Laboratory (NVBL) pooled basic science capabilities to address the COVID-19 pandemic. With $5 Million in initial funding, allocated over the next two years, Brookhaven Lab and its partners will develop a coordinated approach for addressing biosecurity challenges. This pilot study will lead to a roadmap for building out a DOE-wide capability known as the National Virtual Biosecurity for Bioenergy Crops Center (NVBBCC).

“A robust biosecurity capability optimized to respond rapidly to biological threats to bioenergy crops requires an integrated and versatile platform,” said Martin Schoonen, Brookhaven Lab’s Associate Laboratory Director for Environment, Biology, Nuclear Science & Nonproliferation, who will serve as principal investigator for the pilot project. “With this initial funding, we’ll develop a bio-preparedness platform for sampling and detecting threats, predicting how they might propagate, and understanding how pests or pathogens interact with bioenergy crops at the molecular level—all of which are essential for developing short-term control measures and long-term solutions.”

Read more on the Brookhaven National Laboratory website

Image: Pilot study on an important disease in sorghum (above) will develop understanding of threats to bioenergy crops, potentially speeding the development of short-term responses and long-term mitigation strategies

Credit: US Department of Energy Genomic Science Program

#SynchroLightAt75 – APS lights the way to 2012 Chemistry Nobel

#SynchroLightAt75 – APS lights the way to 2012 Chemistry Nobel

Thanks in part to research performed at the U.S. Department of Energy’s (DOE) Argonne National Laboratory, the 2012 Nobel Prize in Chemistry was awarded today to Americans Brian Kobilka and Robert Lefkowitz for their work on G-protein-coupled receptors.

G-protein-coupled receptors, or GPCRs, are a large family of proteins embedded in a cell’s membrane that sense molecules outside the cell and activate a cascade of different cellular processes in response. They constitute key components of how cells interact with their environments and are the target of nearly half of today’s pharmaceuticals.

These medicines work by connecting with many of the 800 or so human GPCRs. But to do this well, a drug needs to connect to the protein like a key opens a lock. Improving drugs requires knowing exactly how these proteins work and are structured, which is difficult because the long, slender protein chains are folded in an intricate pattern that threads in and out of the cell’s membrane.

In a study performed at Argonne in 2007, Kobilka, a professor at Stanford University, used intense X-rays produced by the laboratory’s Advanced Photon Source (APS) to make the first discovery of the structure of a human GPCR. This receptor, called the human β2 adrenoreceptor (β2AR), is responsible for a number of different biological responses, including facilitating breathing and dilating the arteries.

Read more on the Argonne National Laboratory website

Image: This is an image of a G-protein-coupled receptor signaling complex whose structure was identified in 2011. The receptor is in magenta while the different G protein subunits are colored green, red and blue. Stanford biochemist Brian Kobilka shared the 2012 Nobel Prize in Chemistry for his work in determining the structure of this activated GPCR using X-rays provided by Argonne’s Advanced Photon Source.

New insights of how the HIV-1 assembles and incorporates the Env protein

New insights of how the HIV-1 assembles and incorporates the Env protein

Assembly of HIV-1, which causes AIDS, takes place on the inner plasma membrane leaflet of infected cells, a geometric building process that creates hexamers out of trimers of the viral Gag protein, as guided by Gag’s N-terminal matrix domain.

Yet certain details of that virion assembly have been lacking for four decades. In a study published in the journal Proceedings of the National Academy of Sciences of the United States of America, Jamil Saad, Ph.D., University of Alabama at Birmingham (UAB), and colleagues provide the first atomic view of the matrix lattice, a step that advances the understanding of key mechanisms of viral assembly and viral envelope protein incorporation.

“Our findings may facilitate the development of new therapeutic agents that inhibit HIV-1 assembly, envelope incorporation and ultimately virus production,” said Saad, a professor of microbiology at UAB.

The Gag protein is post-translationally modified, in which a lipid-like myristate group is added to help Gag bind to the plasma membrane. How the myristoylated matrix domain, or myrMA, of Gag assembles into lattice eluded detection until now. 

Techniques with low molecular resolution — such as cryo-electron diffraction and cryo-electron tomography — suggested that the myrMA protein organizes as trimers, and these trimers then undergo higher-order organization to form hexamers of trimers. Saad’s study is consistent with a recent study, which suggested that the myrMA protein undergoes dramatic structural changes to allow the formation of distinct hexameric lattices in immature and mature viral particles. Virus maturation is the last step of the virus replication cycle, as the capsid core forms inside the assembled virus, yielding infectious particles.

The envelope protein of HIV-1, or Env, is a transmembrane protein delivered to the plasma membrane by the cell’s secretory pathway. The bulk of the Env protein extends beyond the membrane, but a tail hangs through the membrane back into the inside of the cell. Genetic and biochemical studies have suggested that incorporation of the viral Env protein into the virus particles also depends on interaction between the myrMA domain and the cytoplasmic tail of Env. In 2017, Saad’s lab solved the high-resolution structure of the cytoplasmic tail of Env, which was the last unknown protein structure of HIV-1.

Env is a key infectivity protein. As a mature HIV-1 virus approaches a target cell, Env attaches to proteins on the outside of the uninfected cell, and then the Env protein snaps like a mousetrap to fuse the viral membrane with the cell membrane. 

The structures described by Saad and UAB colleagues showing molecular details at 2.1- angstroms resolution were determined via x-ray diffraction data collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID at the Advance Photon Source. The structures show that the myristic acid of myrMA plays a key role in stabilizing the lattice structure, so the ability to form crystals of myrMA was important. They achieved this elusive technical challenge by removing 20 amino acids from the end of the 132-amino acid myrMA. Formation of a Gag lattice on the plasma membrane is known to be obligatory for the assembly of immature HIV-1 and Env incorporation. 

Read more on the Argonne website

Image: X-ray crystallography revealed the structure of the HIV-1 matrix protein at 2.1 angstroms resolution, advancing understanding of key mechanisms of viral assembly.

Structural studies of SARS-CoV-2 nucleocapsid protein

Structural studies of SARS-CoV-2 nucleocapsid protein

Perspectives in relation to diagnosis and drug design

 A novel zoonotic coronavirus SARS-CoV-2 was originally explored in Wuhan, China in December 2019 and further regarded to the serious pandemic known as COVID-19. In early March 2022, the global COVID-19 pandemic has caused over 453 million confirmed cases and over 6 million deaths (John Hopkins Coronavirus Resource Center, https://coronavirus.jhu.edu).

 The COVID-19 virus and the emergence of new virus variants seriously threat to global public health. It is a strong requirement to develop the effective diagnostic tools which are able to quickly and reliably detect active SARS-CoV-2 infections.

 Structural proteins of the COVID-19 virus are very important to understand its pathogenic mechanism, thus leading to the development of antibodies, vaccines and drugs for targeting these proteins and viruses.

 SARS-CoV-2 comprised the four structural proteins; the spike (S), nucleocapsid (N), envelope (E) proteins and membrane glycoprotein (M). A complete virus particle (virion) is represented in Figure 1. Cryo-electron microscopy is one of the powerful tools to determine the overall structure of the S protein, thus presenting a unique crown or ‘corona’-like shape.

 Three viral proteins; the spike (S), envelope (E) and membrane (M) are embedded in the outer layer of the corona viral particle. The corona viruses protect themselves from the surrounding environment, then the ribonucleic acid (RNA) forms a stable packed in the lipid membrane. The nucleocapsid protein (nucleoprotein) is responsible for tightly wrap the RNA of viruses. However, the fatty membrane of SARS-CoV-2 is sensible to be destroyed by soap, detergent or surfactant.

 The nucleocapsid protein significantly involves in viral genomic RNA binding, thus protecting the coiled RNA as its genetic material inside the virus particle. Moreover, the N protein also plays an important role in the early stages of viral infection when the RNA genome is first released into the target host cell.

 X-ray crystal structures of the N-terminal (PDB entry 7CDZ) and C-terminal domains have been illustrated here (PDB entry 6WZO). Holo structure of N-terminal domain in complex with double strand RNA (PDB entry 7ACS) has been determined by Nuclear Magnetic Resonance Spectroscopy technique.

Read more on the Thai Synchrotron website

Image:  Three dimensional models of the SARS-CoV-2 virion and a schematic diagram of its four structural proteins. 

Credit: Figures were modified from coronavirusexplained     

Structure-guided nanobodies block SARS-CoV-2 infection

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).

New targets for antibodies in the fight against SARS-CoV-2

New targets for antibodies in the fight against SARS-CoV-2

An international team of researchers examined the antibodies from a large cohort of COVID-19 patients. Due to the way antibodies are made, each person that is infected has the potential to produce many antibodies that target the virus in a slightly different way. Furthermore, different people produce a different set of antibodies, so that if we were to analyse the antibodies from many different patients, we would potentially be able to find many different ways to neutralise the virus.

The research article in the journal Cell is one of the most comprehensive studies of its kind so far. It is available online now and will be published in print on 15 April. These new results now show that there are many different opportunities to attack the virus using different antibodies over a much larger area than initially thought/mapped.

Professor Sir Dave Stuart, Life Sciences Director at Diamond and Joint head of Structural Biology at the University of Oxford, said:

SARS CoV-2 is the virus that causes COVID-19. Once infected with this virus, the human immune system begins to fight the virus by producing antibodies. The main target for these antibodies is the spike protein that protrudes from the virus’ spherical surface. The spike is the portion of the virus that interacts with receptors on human cells. This means that if it becomes obstructed by antibodies, then it is less likely that the virus can interact with human cells and cause infection.

By using Diamond Light Source, applying X-ray crystallography and cryo-EM, we were able to visualise and understand antibodies interact with and neutralize the virus. The study narrowed down the 377 antibodies that recognize the spike to focus mainly on 80 of them that bound to the receptor binding domain of the virus, which is where the virus spike docks with human cells.

Read more on the Diamond website

Image: Figure from the publication showing how the receptor binding domain resembles a human torso.

Credit: The authors (Cell DOI: 10.1016/j.cell.2021.02.032)