Highlights from the beamlines – 2015

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How Proteins Organize Themselves

From the LBNL News Release (writer Glenn Roberts): Scientists have for the first time viewed how bacterial proteins self-assemble into thin sheets and begin to form the walls of the outer shell for nano-sized polyhedral compartments that function as specialized factories. The new insight may aid scientists who seek to tap this natural origami by designing novel compartments or using them as scaffolding for new types of nanoscale architectures, such as drug-delivery systems. (Work done at beamlines 5.0.1 and 5.0.2) 

 

Markus Sutter, Matthew Faulkner, Clément Aussignargues, Bradley C. Paasch, Steve Barrett, Cheryl A. Kerfeld, and Lu-Ning Liu, "Visualization of Bacterial Microcompartment Facet Assembly Using High-Speed Atomic Force Microscopy," Nano Letters, DOI: 10.1021/acs.nanolett.5b04259, November (2015).


 

 

Using Bacteria Against Themselves

We think of bacteria as being "all against us", but in fact they fight with each other as much as they invade us. In this study, scientists structurally characterized the proteins forming a bacterial "dagger" which injects toxins into neighboring bacterial cells. The study gives clues as to how we might turn this bacterial aggression system into a tool for fighting bacterial infections. (Work done at beamline 8.2.2)

 

2015_cell_mougous

J.C. Whitney, D. Quentin, S. Sawai, M. LeRoux, B.N. Harding, H.E. Ledvina, B.Q. Tran, H. Robinson, Y. Ah Goo, D.R. Goodlett, S. Raunser, J.D. Mougous, "An Interbacterial NAD(P)+ Glycohydrolase Toxin Requires Elongation Factor Tu for Delivery to Target Cells," Cell 163, 607 (2015).


 

 

The Power of Mold

Forming endoperoxides - molecules containing two consecutive oxygen atoms - requires a very complex chemical reaction, and humans (as well as many other organisms) have special enzymes to produce endoperoxides. Although many of these molecules are very beneficial for human health, and especially important in therapeutics, the mechanism of the reaction had never been fully understood or successfully replicated in the lab. In this study, scientists solved the structure of an enzyme from a common mold, and in the process fully elucidated for the first time the details of the chemical mechanism of endoperoxide formation. 

W. Yan, H. Song, F. Song, Y. Guo, C.-H. Wu, A. S. Her, Y. Pu, S. Wang, N. Naowaronjna, A. Weitz, M.P. Hendrich, C.E. Costello, L. Zhang, P. Liu, Y.J. Zhang,"Endoperoxide formation by an alpha-ketoglutarate-dependent mononuclear non-haem iron enzyme," Nature, Nov 26; 527(7579):539-43, (2015).

 


 

A way to scavenge radioactive material from the body

From the LBNL news story:

Research led by Berkeley Lab’s Rebecca Abergel, working with the Fred Hutchinson Cancer Research Center in Seattle, has found that plutonium, americium, and other actinides can be transported into cells by an antibacterial protein called siderocalin, which is normally involved in sequestering iron.

Their results were published online recently in the journal Proceedings of the National Academy of Sciences in a paper titled, “Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides.” The paper contains several other findings and achievements, including characterization of the first ever protein structures containing transuranic elements and how use of the protein can sensitize the metal’s luminescence, which could lead to potential medical and industrial applications.

Abergel’s group has already developed a compound to sequester actinides and expel them from the body. They have put it in a pill form that can be taken orally, a necessity in the event of radiation exposure amongst a large population. Last year the FDA approved a clinical trial to test the safety of the drug, and they are seeking funding for the tests.

 

The structures of the protein bound to the various radionucelotide samples were solved using beamline 5.0.2.
  Benjamin E. AllredPeter B. RupertStacey S. GaunyDahlia D. AnCorie Y. RalstonManuel Sturzbecher-HoehneRoland K. StrongRebecca J. Abergel, "Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides," PNAS, 112(33) pg 10342 (2015).

 


 

Maintaining lipid balance in cells

The enzyme called SCD1 is responsible for maintaining the balance between saturated and monosaturated fats in cells. When SCD1 stops functioning properly, the body is at a higher risk for metabolic diseases, diabetes, and even cancer. Because of its importance in maintaining healthy cell lipid concentrations, and because high expression of SCD1 is associated with higher insulin and triglyceride levels, many inhibitors of SCD1 have been designed (and patented) in the past few years. In this study, SCD1 was crysallized and its structure solved for the first time in complex with its substrate, the molecule stearoyl-Coenzyme A. The structure shows how the enzyme catalyzes the desaturation of the stearoyl group, and also shows how it anchors to the endoplasmic reticulum in the cell.

H. Wang, M.G. Klein, H. Zou, W. Lane, G. Snell, I. Levin, K. Li, B.C. Sang, "Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate," Nature Structural Molecular Biology, 22(7) pg 581 (2015).

 


 

How Our Brains Tell the Difference Between Dopamine and Cocaine

We have specialized transporter proteins in our neural synapses which respond to neurotransmitters - chemicals such as dopamine - which can affect our mood and contribute to neural development and function. Unfortunately those same proteins also respond to chemicals such as cocaine and methamphetamine which are not endogenous to our bodies. Transporter proteins are notoriously difficult to crystallize, and so we do not have very much information on their structure and corresponding functioning. In this study, the Gouaux lab managed to obtain atomic-level structures of one of the dopamine transporter proteins, both with and without several neurotransmitters bound to it. At first glance, the structure shows that the binding pocket of the protein, as expected, fits the dopamine neurotransmitter very well. However, the structures also showed that the protein can "remodel" itself to bind other differently shaped chemicals such as methamphetamine and cocaine. This observed plasticity of the binding site supports the "induced-fit" theory of ligand binding, in which proteins change their structure to accommodate their binding partners, as opposed to the "lock-and-key model" in which protein structure is rigid and perfectly matched (lock) to the ligand (key). 

K. Wang, A. Penmatsa, E. Gouaux,"Neurotransmitter and psychostimulant recognition by the dopamine transporter," Nature, Vol. 521, May 21, pg 322 (2015).

 
 

A glimpse into the code that controls variety of cell functions

From NIH news, Tues May 12 2015: Microtubules are cylindrical structures that provide shape to cells and act as conveyor belts, ferrying molecular cargo throughout cells. Although all microtubules have the same basic appearance, they are marked on their outside surface with a variety of chemical groups. These markers impact a cell’s activity by changing the stability of microtubules, thus affecting cell shape, or by repositioning molecular cargo traveling on the microtubules.

“The microtubule markers are constantly being added and removed, depending on the local needs of the cell. Think about a highway system where street signs are constantly changing and roads are quickly built or torn apart,” said Dr. Roll-Mecak, Ph.D., NINDS scientist and senior author of the study. The most common microtubule marker in the brain is glutamate. The addition of glutamate markers to microtubules plays important roles in brain development and brain cell repair following injury. For example, one of the signatures of damaged cells in cancer or blunt trauma is a change in the pattern of these microtubule markers. In addition, mutations in TTLL genes have been linked with several neurodegenerative disorders. This research may lead to the development of small molecules that can regulate activity of TTLL proteins, which may have implications for disorders linked to mutations in TTLL genes.    

C.P. Garnham, A. Vemu, E.M. Wilson-Kubalek, I. Yu, A. Szyk, G.C. Lander, R.A. Milligan, A. Roll-Mecak,  “Multivalent Microtubule Recognition by Tubulin Tyrosine Ligase-Like Family Glutamylases,” Cell. May 7, (2015).


 

How the drug Zydelig works to treat leukemia

The drug Zydelig (also known as Idelalisib) was recently approved in both the United States and the European Union for patients whose chronic lymphocytic leukemia has relapsed, and as a first-line therapeutic against  certain other lymphomas. It was known that the drug works to inhibit the cellular process of converting PIP2 to PIP3, and as such can inhibit the activation of certain pathological cellular pathways that are common in hematological malignancies. What wasn't known until recently was how the drug binds to and inhibits its target. This study by Gilead Sciences, using beamlines 5.0.1 and 5.0.2, pinpoints how Zydelig is both potent and selective, and points the way to developing even more effective drugs.

J.R. Somoza, D. Koditek, A.G. Villaseñor, N. Novikov, M.H. Wong, A. Liclican, W. Xing, L. Lagpacan, R. Wang, B.E. Schultz, G.A. Papalia, D. Samuel, L. Lad, and M.E. McGrath, "Structural, Biochemical, and Biophysical Characterization of Idelalisib Binding to Phosphoinositide 3-Kinase delta," J. Biol. Chem, V290, No. 13, p8439 (2015).

 


 

Watching Neural Circuitry in Action

Neuroscientists now have a method to mark active populations of neurons in vivo to study circuit activity in the behaving animal. Fosque et al. designed and thoroughly validated via crystallographic studies a fluorescent protein–based reagent that allows permanent marking of active cells over short time scales. This indicator, termed CaMPARI, switches from its native green to a red fluorescent state by simultaneous illumination with violet light and exposure to increased levels of intracellular calcium. CaMPARI successfully marked active nerve cells in Drosophila, zebrafish, and mouse brains. B.F. Fosque, Y. Sun, H. Dana, C.-T. Yang, T. Ohyama,M.R. Tadross, R. Patel, M. Zlatic, D.S. Kim, M.B. Ahrens, V. Jayaraman, L.L. Looger, E.R. Schreiter, "Labeling of active neural circuits in vivo with designed calcium integrators," Science, Vol. 347, Feb 13, no. 6223, pg 755 (2015).

 


 

Designing a Genetic Firewall into GMOs

One of the biggest fears of GMO foods is based on the possibility that they can escape into the natural ecosystem. Containment is tricky because GMOs, even if they are designed with an internal "kill-switch", as they often are, can mutate out that kill-switch when faced with evolutionary pressure to survive. The researchers in this study came up with an alternative method for containing GMO organisms: design a NSAA (non-standard amino acid) into the normal sequence of several of the proteins that the GMO relies on. If the organism does not have access to the NSAA, it cannot survive. This study showed that such an engineered protein was not only completely dependent on the NSAA for survival, but that it was not able to mutate out the change leading to the NSAA. Crystallographic analysis of the proteins at the BCSB beamlines demonstrated that their structures were as predicted. 

D.J. Mandell, M.J. Lajoie, M.T. Mee, R. Takeuchi , G. Kuznetsov , J.E. Norville , C.J. Gregg, B.L. Stoddard & G.M. Church, "Biocontainment of genetically modified organisms by synthetic protein design," Nature, Vol 518, Feb 5, pg 55 (2015).    

 


 

Using Ancient Protein Structure to Uncover Modern Drug Mechanisms

Tiny differences in a protein's sequence can make a huge difference in how well the protein binds drugs. One example is the structure of the drug Gleevec bound to its target kinase, Abl. Gleevec does not bind nearly as well to the kinase Src, which is nearly identical in structure to Abl. To get at what causes this difference, Kern's group reconstructed the evolutionary changes in the two kinases, and found that mutations that affected conformational dynamics are at the root of the answer. The ligand binding pocket reorganizes itself in response to drug binding (the "induced-fit equilibrium" model of ligand binding) and certain mutations cause this induced-fit to work better. The kinase Abl binding Gleevec is one of these cases, and explains why Gleevec is so effective in treating multiple cancers: the Abl kinase is active in cancer cells and not in healthy cells, so Gleevec is extremely well targeted. 

C. Wilson, R. V. Agafonov, M. Hoemberger, S. Kutter, A. Zorba, J. Halpin, V. Buosi, R. Otten, D. Waterman, D. L. Theobald,  D. Kern, "Using ancient protein kinases to unravel a modern cancer drug’s mechanism," Science, Vol 347, Issue 6224, pg 882 (2015).