#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

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

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

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

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)

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

Newly discovered photosynthesis enzyme yields evolutionary clues

Rubisco is one of the oldest carbon-fixing enzymes on the planet, taking CO2 from the atmosphere and fixing it into sugar for plants and other photosynthetic organisms. Form I (“form one”) rubisco goes back nearly 2.4 billion years and is a key focus of scientists studying the evolution of life as well as those seeking to develop bio-based fuels and renewable-energy technologies. A newly discovered form of rubisco—dubbed form I′ (“one prime”)—is thought to represent a missing link in the evolution of photosynthetic organisms, potentially providing clues as to how this enzyme changed the planet.

To learn how form I′ rubisco compares to other rubisco enzymes, researchers performed x-ray crystallography at Advanced Light Source (ALS) Beamline 8.2.2. Then, to capture how the enzyme’s structure changes during different states of activity, they applied small-angle x-ray scattering (SAXS) using Beamline 12.3.1 (SIBYLS). This combination of approaches enables scientists to construct unprecedented models of complex molecules as they appear in nature.

Read more on the ALS website

Image: A ribbon diagram (left) and molecular surface representation (right) of carbon-fixing form I′ rubisco, showing eight molecular subunits without the small subunits found in other forms of rubisco. An x-ray diffraction pattern of the enzyme, also generated by the research team, is in the background.

Credit: Henrique Pereira/Berkeley Lab

Diamond helps uncover how an untreatable cancer-causing virus affects immune cells

Scientists have found that human T-cell lymphotropic virus, type 1 (HTLV-1) hijacks cellular machinery to establish an infection.  

Research was undertaken using cutting-edge visualisation techniques such as X-ray crystallography, which was undertaken at Diamond, and single-particle cryo-electron microscopy (cryo-EM).  

HTLV-1 is a virus that affects T cells, a type of white blood cell which plays a crucial role in our immune system. Currently, between five and 20 million people worldwide are infected by HTLV-1 and no cure or treatment is available. While most people infected with the virus do not experience symptoms, around two to five per cent will go on to develop adult T-cell leukaemia (ATL).  

New research, led by a team from Imperial College London and the Francis Crick Institute, shows in atomic detail how HTLV-1 infects immune cells. By providing a more nuanced understanding of how the virus establishes infection in the body, the research will help to support the development of new, targeted therapies. 

Read more on the Diamond Light Source website

Image: Scanning electron micrograph of a human T lymphocyte (also called a T cell) from the immune system of a healthy donor. Credit: NIAID