Recent highlights from the beamlines

 

Coronavirus-mediated pandemics are a serious treat to humans, as observed in outbreaks of SARS and MERS. The work described in this paper provides a structural framework for understanding the mechanism of action from survivors’ neutralizing antibodies.

A.C. Walls, X. Xiong, Y.-J. Park, M.A. Tortorici, J. Snijder, J. Quispe, E. Cameroni, R. Gopal, M. Dai, A. Lanzavecchia, M. Zambon, F.A. Rey, D. Corti, and D. Veesler, “Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion,” Cell, doi:10.1016/j.cell.2018.12.028 (2019).

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From the LBNL site here:

Photosynthesis is the process by which plants use water and sunlight to produce the carbohydrate molecules that sustain life on Earth. The molecular rearrangements involved in this process have been very difficult to study, because it is a kinetic process involving the subtle repositioning of atoms and the directed flow of electrons from one molecule to another. Now, using both synchrotron diffraction data, and data obtained at the LCLS (a free electron laser source), these researchers were able to delineate six distinct steps in the molecular movements that are part of the photosynthesis cycle.

 

First frames of a movie of the water oxidation reaction in nature: Light-induced changes observed at the Mn cluster of photosystem II as it goes through its catalytic cycle. (Credit: Berkeley Lab)

Kern et al “Structures of the intermediates of Kok’s photosynthetic water oxidation clock,” Nature Vol 563, pages 421–425 (2018).

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The NSP2 protein forms an octomer (the monomer is shown in cyan.) This protein is one of several that form the lattice making up the “virus factory.”

Many DNA and RNA virus pathogens require a lattice-like structure in order to replicate. This lattice is called a viroplasm and is composed of both virus particles and proteins. In this study, researchers found that interactions between certain nonstructural proteins and enzymes trigger (through phosphorylation) the formation of the lattice structure. Similar mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.

J.M. Criglar, R. Anish, Liya Hu, S.E. Crawford, B. Sankaran, B.V.V. Prasad, and M.K. Estes, “Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories,” PNAS, doi:10.1073/pnas.1717944115 (2018).

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We normally think of bacteria in their relationship with us: the “good guys” help us by living synergistically inside us and promoting a healthy gut, for instance, while the “bad guys” can give us infections and weaken or kill us. But one thing we don’t often realize is that bacteria are constantly fighting each other as well. This study revealed a previously uncharacterized toxin that is used by certain bacteria to kill of their bacterial neighbors. The study also showed exactly how the “attacking” bacteria protects itself against its own deadly weapon. One very interesting finding in the study was that the biochemical method by which the toxin acts is very similar to the mechanism of toxins produced in human diseases such as cholera and diphtheria, even though those human diseases emerged much later in evolution.

Healthy bacteria (left) and bacteria (right) whose cell-division machinery has been disrupted by a toxin newly discovered in some bacterial arsenals. (Image credit: Mougous Lab) and featured on the LBNL Biosciences page here.

Ting et al, “Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins,” Cell, Vol 175, No5, P1380-1392.E14, Nov 15, 2018.

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Designing cancer drugs can be tricky because of the “off-targets”: the fact that drugs will often bind to more than their intended target. Recently, a promising new cancer drug, SHP099, was developed to target proteins that have been mutated in defective cells and lead to cancers such as juvenile myelomonocytic leukemia. In this study, researchers determined that the drug SHP099 acts through allosteric inhibition, which means inhibiting a protein by binding at a position other than its active site. This form of inhibition is much more selective than binding to active sites, since allostery can be unique to a signaling pathway. The research opens the door for development of an entirely new class of cancer drugs.

The drug SHP009 (colored red, blue, green) binding to its target protein.

R.A.P. Pádua, Y. Sun, I. Marko, W. Pitsawong, J.B. Stiller, R. Otten and D. Kern, “Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2,” Nature Communications, 9, Article number: 4507 (2018).

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The “beta barrel” protein that was designed at the Institute for Protein Design. One end of the barrel is designed to stabilize the protein, and the other end creates a cavity that can bind the target molecule.

From the University of Washington news release:

For the first time, scientists have created, entirely from scratch,  a protein capable of binding to a small target molecule.

Previously, such small-molecule binding proteins have been made by altering proteins already existing in nature.  That approach  significantly limited the possibilities. The ability to make such proteins from scratch, or “de novo,” opens the way for scientists to create proteins unlike any found in nature. These proteins can be custom-designed with high precision and affinity to bind to and act on specific small molecule targets.

Jiayi Dou, Anastassia A. Vorobieva, William Sheffler, Lindsey A. Doyle, Hahnbeom Park, Matthew J. Bick, Binchen Mao, Glenna W. Foight, Min Yen Lee, Lauren A. Gagnon, Lauren Carter, Banumathi Sankaran, Sergey Ovchinnikov, Enrique Marcos, Po-Ssu Huang, Joshua C. Vaughan, Barry L. Stoddard & David Baker, “De novo design of a fluorescence-activating β-barrel,” Nature 561, 485 (2018).

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A helicase protein in action unwinding a DNA quadruplex. 

DNA doesn’t just form the classic double-helix; it can also form quadruplexes, composed of two helices. The cell has specialized mechanisms using protein helicases to unwind this form of DNA when needed. The researchers in this study captured one of the steps during unwinding using crystallography. You can read more details at the ALS Science Briefs page here.

M.C. Chen, R. Tippana, N.A. Demeshkina, P. Murat, S. Balasubramanian, S. Myong, and A.R. Ferré-D’Amaré, “Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36,” Nature 558, 465 (2018).

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Researchers used the inherent programmability  of DNA to create “designer” DNA lattices: three dimensional arrays of DNA in specific arrangements. The interactions between strands of DNA were controlled via Watson-Crick pairing, while the higher-order symmetry of the array was organized into repeating Holiday junctions. The result was a crystal of DNA of predicted symmetry and packing, providing a lattice with periodic cavities that could be used to hold proteins or other organic or inorganic materials. These programmed lattices are of great interest in a number of applications, from immobilizing enzymes for catalysis reactions to drug delivery systems. 

Representation of the six layers of DNA strands which make up the lattice. 

 

F. Zhang, C.R. Simmons, J. Gates, Y. Liu, H. Yan, “Self-Assembly of a 3D DNA Crystal Structure with Rationally Designed Six-Fold Symmetry” Angew. Chem. 57, 12504-12507 (2018).

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Adapted from Figure 4 of the paper, showing the binding sites on interleukin-2 for its receptor. 

 

 

Cytokines are proteins released by cells in order to influence the cells around them. Our immune systems release a particular type of cytokine, called a Interleukin-2, in order to regulate our immune response to perceived danger. This is useful to keep our immune response in check, because sometimes, as in the case of autoimmune diseases, the immune system will launch too great a response and end up doing more harm than good. Interleukin-2, therefore, is a potential therapy against autoimmune diseases. 

Trotta et al, “A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism” Nature Medicine  (2018).

From the ALS website:

Our daily function depends on signals traveling between nerve cells (neurons) along fine-tuned pathways. Central nervous system neurons contain acid-sensing ion channel 1a, a protein important in sensing pain and forming memories of fear. This channel opens and closes in response to changes in extracellular proton concentrations. When protons accumulate outside the neuron, the channel opens, allowing sodium ions to flow into the cell, depolarizing the cell membrane and generating an electrical signal. The channel eventually becomes desensitized to protons and the gate closes. Scientists have visualized both the open and desensitized channel structures, but the third structure, which forms when the protons dissipate and the channel closes, remained elusive until now.

This gif is from the ALS website. It depicts the opening and closing of the protein channel, which changes as a function of proton concentration.

 

N. Yoder, C. Yoshioka, and E. Gouaux, “Gating mechanisms of acid-sensing ion channels,”Nature 555, 397–401 (2018), doi:10.1038/nature25782.

 

 

Some inherited muscle diseases are characterized by periodic muscle paralysis. These diseases result from mutation of specific residues in sodium-channel proteins, which are responsible for controlling salt intake into cells and are the basis for maintaining voltage potentials which enable muscle contractions. In this study, the researchers used structural data on mutated and wild-type sodium channels to provide direct evidence for the “salt leakage” theory of how these channel proteins allow sodium ions back through the channels at the wrong time.

D. Jiang, T.M.G. El-Din, C. Ing, P. Lu, R. Pomes, N. Zheng, W.A. Catterall, “Structural basis for gating pore current in periodic paralysis.” Nature, 557, 590-594 (2018).

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Adapted from Figure 6b. F- ions are magenta, and H+ ions are blue. A glutamate residue changes rotameric conformation during the transport cycle. 

 

 

Fluoride in the environment can be toxic to organisms, but some bacteria have developed specialized membrane channel proteins to expel fluoride. This mechanism of transporting fluoride very selectively without transporting chloride, which is much more abundant in the environment, has until recently been unknown. Using structures of several fluoride transporters, this study reveals a possible “windmill” mechanism of transport, in which a specific residue alternately flips conformation in order to bind or expel a fluoride ion. 

“A CLC-type F-/H+ antiporter in ion-swapped conformations,” Nat. Struct. Mol. Biol. June, 2018. N.B. Last, R.B. Stickbridge, A.E. Wilson, T. Shane, L. Kolmakova-Partensky, A. Koide, S. Koide, C. Miller. https://doi.org/10.1038/s41594-018-0082-0

 

 

This research isolated human-derived antibodies and used them to confer protection against malaria in mice. The structural studies of the antibody at beamlines 5.0.1 and 5.0.2 showed how the antibody worked, and how to adapt it into a vaccine for humans. See also News & Views: “A new site of attack for a malaria vaccine.” (March 19, 2018)

N.K. Kisalu et al, “A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite,” Nature Medicine 24(4), p.408. April 2018. doi:10.1038/nm.4512

 

Surface representation of the antigen-binding fragment (light chain in tan and heavy chain in light blue) in complex with relevant peptide based on the obtained crystal structures. 

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