Designing better cholesterol-lowering Drugs
From the ALS site:
"The most commonly prescribed cholesterol-lowering drugs, called statins, are effective but associated with a number of side effects, including muscle pain and digestive problems. Genentech has been working in collaboration with the ALS with the goal of identifying a better treatment mechanism that targets a cholesterol-regulating protein in the body known as PCSK9. Recent advances in understanding PCSK9’s structure have put them closer to that goal."
Y. Zhang, M. Ultsch, N. Skelton, S. Burdick, M. Beresini, W. Li, M. Kong-Beltran, A. Peterson, J. Quinn, C. Chiu, Y. Wu, S. Shia, P. Moran, P. Di Lello, C. Eigenbrot, and D. Kirchhofer, “Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists,” Nat. Struct. Mol. Biol. 24, 848 (2017). doi: 10.1038/nsmb.3453
Structure of the Fc tail domain of the modified IgG antibody. An extreme twist in the second homodimer (on the right) accounts for its selectivity in triggering one particular immune-system pathway.
From the ALS site:
"Immunotherapy—the use of the immune system to fight disease—has made tremendous progress in the fight against cancer. Antibodies such as immunoglobulin G (IgG) can identify and attack foreign or abnormal substances, including tumor cells. But to control and amplify this response, scientists need to know more about how elements of the immune system recognize tumor cells and trigger their destruction.
In this study, researchers designed an antibody such that it would bind exclusively to a particular protein in an immune pathway, but not affect a similar pathway. The crystal structures revealed out the antibody exhibits such precise selectivity."
C.-H. Lee, G. Romain, W. Yan, M. Watanabe, W. Charab, B. Todorova, J. Lee, K. Triplett, M. Donkor, O.I. Lungu, A. Lux, N. Marshall, M.A. Lindorfer, O.-L. Goff, B. Balbino, T.H. Kang, H. Tanno, G. Delidakis, C. Alford, R.P. Taylor, F. Nimmerjahn, N. Varadarajan, P. Bruhns, Y.J. Zhang, and G. Georgiou, “IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions,” Nat. Immunol. 18, 889 (2017), doi:10.1038/ni.3770.
Disabling a biological weapon
From the Duke press release:
F. tularensis is an exceptionally hardy organism that can infect a variety of hosts, including humans, rabbits and mosquitos, and can survive for weeks at a time in dead and decaying carcasses. It is so virulent that a person only has to inhale 10 microscopic particles of the bacterium to become infected. The Russians and Japanese, as well as the Americans and their allies, all explored its potential as a biological weapon during World War II.
The scientists in this study recently mapped out the complex molecular circuitry that enables F. tularensis to become virulent. The map reveals a unique characteristic of the bacteria that could become the target of future drug development.
“Dissection of the molecular circuitry controlling virulence in Francisella tularensis,” Bonnie Cuthbert, Wilma Ross, Amy Rohlfing, Simon Dove, Richard Gourse, Richard G. Brennan, and Maria A. Schumacher. Genes & Development, September 13, 2017. DOI: 10.1101/gad.303701.117. Link to article.
Deciphering early photosynthesis
Researchers at Arizona State University solved the structure of a membrane protein at the heart of the photosynthetic center from the bacterium Heliobacterium modestricaldum, which is the simplest known bacterium able to drive photosynthesis and is found in muddy soils near hot springs. In contrast to photosynthesis in plants, these bacteria grow without oxygen and use hydrogen sulfide in place of water, and use near-infrared wavelengths as opposed to visible light. The photosynthesis apparatus in these bacteria is thought to represent the earliest common ancestor of all photosynthesis complexes in plants and bacteria, and may have evolved around three billion years ago when the Earth's atmosphere did not contain much oxygen but the oceans contained plenty of hydrogen sulfide. The structure therefore allows a fundamental understanding of the early evolution of photosynthesis. This work also represented a tour de force in crystallography; the researchers first started crystallization attempts on the complex seven years ago, and finally now have a high resolution (2.2A) structure.
Overall structure of the Reaction Center within Heliobacterium modesticaldum. The core polypeptide dimer (red and pink) and two small subunits (orange) coordinate 54 (bacterio)chlorophylls, 2 carotenoids (green), and an FeS cluster (yellow) that capture and transfer energy to the core.
Designing even better self-assembling DNA matrices
The goal of "DNA nanotechnology" - designing self-assembling DNA molecules - is to provide a biological matrix in which other biological "guest" molecules can be trapped in a regular repeating pattern. This would facilitate solving the structures of the guest molecules, which might otherwise be recalcitrant to crystallization on their own. The first self-assembled DNA crystal was reported in 2009. Just last year, Simmons et all reported on a new DNA motif containing an arrangement of continuous helical layers. This new study extends the previous study and presents a better design for holding guest molecules.
Crystals of both B-DNA (rod-shaped), the naturally occurring form of DNA, and L-DNA (square-shaped), which has the opposite handedness. Both types were used to design the DNA matrix described in this paper.
Investigating the tolerance of signalling molecules to mutational change
Proteins tend to be tolerant to changes in their sequence. This is good news for organisms, because random mutations occur regularly but the proteins affected can still fold correctly into their three dimensional structure and retain their proper function. Ras proteins, however, have been puzzling to scientists because they show remarkable conservation in their sequences even across different species, which implies that they are not tolerant to mutation. Ras proteins are signalling proteins important in numerous cellular processes, and are nearly identical in all vertebrates. In this research study, scientists used a combination of structural, biochemical, and functional studies to answer the question of why these signalling proteins have remained conserved throughout evolution. They found that sequence conservation in Ras depends very strongly on the interaction of Ras with its regulators. In other words, it is not necessarily the sequence of Ras alone that is intolerant to mutation, it is the biochemical network in which Ras operates that is intolerant to change. This has important implications for cancer drugs which are being developed to target Ras; as the cancer cells quickly evolve resistance to the drugs, mapping the sensitivity of Ras to mutation within its biochemical network will guide design of better drugs.
Two molecules of Ras bound to its activator protein SOS. Switch I and II represent regions of Ras that change structurally when GTP is converted to GDP. The variable regions are identified as sequence divergence between vertebrates and invertebrates.
How bacteria assemble microcompartments within themselves
From the LBNL science highlights. Full highlight: http://newscenter.lbl.gov/2017/06/22/bacterial-microcompartment-shell/
Some bacteria assemble compartments inside themselves in order to provide a protected environment for certain processes, such as carbon fixation. The compartments, or shells, keep the proteins involved in the process together (sort of like forcing negotiators into a closed room together) in order to speed up the process. They are also used to sequester toxic compounds and keep the rest of the cell safe. The Kerfeld group managed to solved the structure of an intact shell, which is itself made up of hundreds of proteins that have to assemble into a large spherical structure. Her group solved the structure of several of these shell proteins at ALS beamlines 5.0.2 and 5.0.3, and then used cryo electron microscopy to solve the entire intact structure, yielding for the first time a complete structural picture of a bacterial microcompartment.
The entire bacterial shell is large by cellular standards, at about 400 Angstrom across, or about 0.04 microns. Five distinct proteins (hundreds of copies each) line up in a repeating pattern to form the shell. The subassemblies of these proteins form patterns of their own, in hexamers, pentamers, and different types of trimers.
Markus Sutter, Basil Greber, Clement Aussignargues, Cheryl A. Kerfeld, " Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell," Science, 356, 1293–1297 (2017).
Targeting a protein used by ZIka virus for replication
From the ALS science highlights. Full highlight: https://als.lbl.gov/bending-beta-sheet-curve-shape-protein-cavities
Zika virus is a mosquito-borne infectious disease linked to certain birth defects in infants in South and Central America and the United States. A Lawrence Berkeley National Laboratory (Berkeley Lab) researcher, Banumathi Sankaran, worked as part of a multi-institutional team to map a key viral protein called NS5. Necessary to virus reproduction, NS5 contains two enzyme activities: one reduces the body’s ability to mount an immune response against infection and the other helps start the genetic replication process. The work was led by Indiana University’s Cheng Kao and Pingwei Li at Texas A&M University (TAMU).
In a study published March 27 in Nature Communications, the team described the structure and function of these two enzyme active sites. They also showed comparisons between this protein and those from other related viruses that cause dengue fever, West Nile virus, Japanese encephalitis virus, and hepatitis C. These comparisons will help researchers as they search for possible compounds to halt the ability of the virus to reproduce.
Baoyu Zhao, Guanghui Yi, Fenglei Du, Yin-Chih Chuang, Robert C. Vaughan, Banumathi Sankaran, C. Cheng Kao, and Pingwei Li, "Structure and function of the Zika virus full-length NS5 protein," Nat. Commun. 8, 14762 (2017).
Illustration of how a curved protein β sheet (yellow-gold) forms a pocket (gray) to poten-tially fit molecules. Top: Struc-ture based on atomic-resolution data. Bottom: Structure based on a computational model. (Credit: Benjamin Basanta)
E. Marcos, B. Basanta, T.M. Chidyausiku, Y. Tang, G. Oberdorfer, G. Liu, G.V.T. Swapna, R. Guan, D.-A. Silva, J. Dou, J.H. Pereira, R. Xiao, B. Sankaran, P.H. Zwart, G.T. Montelione, and D. Baker, “Principles for designing proteins with cavities formed by curved β sheets,” Science 355, 201 (2017). doi:10.1126/science.aah7389
Shaping protein surfaces and cavities
From the ALS science highlights. Full highlight: https://als.lbl.gov/bending-beta-sheet-curve-shape-protein-cavities
Curved β sheets are basic building blocks in the construction of protein cavities that, by serving as binding sites for other molecules, are essential to protein function. After analyzing classic protein formations and running folding simulations, researchers designed a series of novel proteins with curved β sheets, inspired by naturally occurring protein superfamilies. They then compared the predicted models to physical examples of these designed proteins using x-ray crystallography. All of the structures closely matched the predicted models, showing that β-sheet curvature can be controlled with atomic-level accuracy. The discovery opens the door to the design of new proteins capable of entirely new functions, including catalyzing reactions not seen in nature and the development of new diagnostic tests and medical treatments.
Deciphering enzymes that breakdown biofuel waste
From the Berkeley Today article on April 5, 2017:
"A protein used by common soil bacteria is providing new clues in the effort to convert aryl compounds, a common waste product from industrial and agricultural practices, into something of value. In biofuel production, aryl compounds are a byproduct of the breakdown of lignin. Many of the pathways leading to the breakdown of lignin involve demethylation, which is often a critical precursor to any additional steps in modifying lignin-derived aryl compounds. The researchers found that half of the LigM enzyme was homologous to known structures with a tetrahydrofolate-binding domain that is found in simple and complex organisms alike. The other half of LigM’s structure is completely unique, providing a starting point for determining where its aryl substrate-binding site is located. They also figured out that LigM is a tyrosine-dependent demethylase. The researchers are now working on engineering LigM so that it is able to act on a wider range of aryl substrates in addition to targeting specific aryl waste products."
The crystalographic structure of LigM. Novel structural elements that are unique to LigM are in red, and a conserved tetrahydrofolate-binding domain in gray, and substrates (green).
Amanda C. Kohler, Matthew J. L. Mills, Paul D. Adams, Blake A. Simmons, Kenneth L. Sale, "Structure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism," PNAS, 2017, April 5. doi: 10.1073/pnas.1619263114
Using DNA as an Engineering Tool
Using DNA in nanotechnology applications has gained interest in recent years because it is a "highly programmable polymer." DNA can be precisely controlled as a building block by modifying the sequence in order to control affinity through base-pairing, and create 2d and 3d self-assembling matrices. The resulting matrices in turn serve as platforms to precisely control chemical processes or to organize other macromolecules such as proteins and viruses. In this study, researchers designed an entirely new DNA motif which self-assembles into a regular 3d lattice.
View of the crystal structure of the 3d DNA assembly. White brackets indicate the stacked helices contained within any given unit cell.
C.R. Simmons, F. Zhang, J.J. Birktoft, X. Qi, D. Han, Y. Liu, R. Sha, H.O. Abdallah, C. Hernandez, Y.P. Ohayon, N.C. Seeman, H. Yan, "Construction and Structure Determination of a Three-Dimensional DNA Crystal," J. Am. Chem. Soc., 2016, 138 (31), pp 10047–10054.
The ALLINI-bound HIV-1 integrase polymer observed in crystals. Bound GSK1264 is shown by the red spheres, two interacting dimers are highlighted in the dotted box, and additional subunits of the open polymer are shown in light grey.
New Drugs To Fight HIV
Designing drugs to fight viral infections is a constant battle, since viruses are constantly evolving and developing drug-resistance. For HIV, a new promising class of antivirals has recently been developed: they are called “ALLINIs” (allosteric inhibitors of integrase) and work by inhibiting HIV replication by inducing aggregation in the late stages of viral particle assembly. This class of drugs is highly potent against HIV replication in cell culture, and so any information about how they function is important for further developing the drugs for use in humans. This study verified that the drugs interfere specifically with HIV-1 integrase, and revealed for the first time the structural basis for how ALLINIs interact with the integrase to cause aggregation. The crystal structures of HIV-1 integrase bound to the ALLINI drug GSK1264 showed the entire ALLINI-binding interface, revealing that the drug catalyzes formation of an open polymer form of the integrase, which interferes with viral particle formation. Diffraction data for the crystal structures was collected at beamlines 5.0.2 and 12.3.1.
Designing Better Antidepressants
Humans have millions of brain cells – neurons - which are in constant communication with each other. This transmission of information is accomplished using neurotransmitters, which are chemicals passed between brain cells, influencing human behavior, cognition, and physiology. One of those chemicals, serotonin, has more than its share of influence, from mood to cardiovascular function, digestion to reproduction. Serotonin is found in the central nervous system and GI tract as well as the brain, though the brain is where it is has a major influence on mood and sense of well-being. The two antidepressants studied here lock SERT in an outward-open configuration by physically binding to the central binding site within the vestibule and precluding further motion. Interestingly, (S)-citalopram was also shown to bind to a site directly adjacent to the central binding site. This study revealed critical details on how and where drugs bind to the serotonin transport proteins, laying the groundwork for the design of new antidepressants.
J.A. Coleman, E.M. Green, E. Gouaux, "X-ray structures and mechanism of the human serotonin transporter," Nature, vol 532, no.7599, 334-339, 2016. (doi: doi:10.1038/nature17629)
Blocking Ebola and Related Filoviruses
From the Stanford Linear Accelerator Center (SLAC) news page: "...scientists have determined in atomic detail how a potential drug molecule fits into and blocks a channel in cell membranes that Ebola and related “filoviruses” need to infect victims’ cells. The study by researchers at University of California, San Francisco marks an important step toward finding a cure for Ebola and other diseases that depend on the channel. 'There are no effective treatments for filovirus infections in humans,' said UCSF postdoctoral researcher Alex Kintzer, who performed the study with Professor Robert Stroud. 'With these new structures, pharmaceutical chemists can now design new candidate drug molecules that would be more efficient and effective in blocking the channel and defeating these viruses.'" To determine the structures, Kintzer first made crystals containing many copies of the target channel protein, called TPC1, bound to the potential drug molecule, Ned-19...the project involved testing about 6,900 crystals during more than 36 sessions at SSRL and ALS. It took nearly four years to complete, from planning to publication." (Work done at SLAC 12-2, and ALS 5.0.2 and 8.3.1)
Tearing Apart the Hendra Virus
The Hendra virus (HeV) infects both horses and humans with a high mortality rate. In this study, scientists elucidated one of the Hendra virus's fusion proteins, which allows the virus to enter host cells. The structure is very similar to the parinfluenza virus 5, which suggest a common mode of action, and providing a starting point for an effective vaccine. (Work done at beamline 8.2.2)
J. Wong, R.G. Paterson, R.A. Lamb, T.S. Jardetzky, "Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form," PNAS, vol 113, no.4, 1056-1061, January 2016. (doi: 10.1073/pnas.1523303113)
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)
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.
Benjamin E. Allred, Peter B. Rupert, Stacey S. Gauny, Dahlia D. An, Corie Y. Ralston, Manuel Sturzbecher-Hoehne, Roland K. Strong, Rebecca J. Abergel, "Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides," PNAS, 112(33) pg 10342 (2015).
Maintaining lipid balance in cells
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
“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
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).
Accelerating Drug Design for Ovarian and Prostate Cancer
Cancer cells often overexpress and release certain proteins that help the cancer spread. The protein PRK1 is one such protein expressed in prostate and ovarian cancer. Because of this, PRK1 is a target for therapeutics for these cancers, and indeed several inhibitors to PRK1 are in clinical trials. This study by researchers at Celgene, using crystal structures solved at beamlines 5.0.1, 5.0.2, and 5.0.3, elucidated the structure of PRK1 both with and without these inhibitors, showing some interesting structural features that will accelerate the design of even more potent and selective drugs to target PRK1.
P. Chamberlain, S. Delker, B. Pagarigan, A. Mahmoudi, P. Jackson, M. Abbasian, J. Muir, N. Raheja, B. Cathers, "Crystal Structures of PRK 1 in Complex with the Clinical Compounds Lestaurtinib and Tofacitinib Reveal Ligand Induced Conformational Changes," PLOS ONE August (2014).
Targeting the Kinase
Protein kinases are used in a huge number of cellular processes. By catalyzing the transfer of a phosphate group, they control cell signalling and influence cell proliferation, cell adhesion, and survival, among a number of other physiological important processes. One drug that has been developed to target kinases is the cancer drug Ruxolitinib, which is a kinase inhibitor and therefore can limit cell proliferation. It has been approved for the treatment of the myeloproliferative neoplasms. But how does it work? In this study, scientists determined the crystal structure of a kinase domain in complex of Ruxolitinib at a high enough resolution to show some of the exact chemical changes that the drug induces. In addition, the study indicates how the drug can me modified to be even more effective.
Y. Duan, L. Chen, Y. Chen, X.G. Fan, "c-Src Binds to the Cancer Drug Ruxolitinib with an Active Conformation," PLOS ONE Sept 8 (2014).
Why Some Cells Should Die
Cells go through a natural process called apoptosis when they are damaged or can no longer function properly. By taking themselves out of commission they reduce the danger to the organism when, for instance, they are infected with a virus. But this process of apoptosis is not advantageous for a virus, which infects a cell and then forces the cell to make more copies of itself, as is the case with the Epstein-Barr virus (EBV). The EBV actually produces inhibitor proteins to counteract the process of apoptosis, keeping the host cell alive long enough to infect other cells. The scientists in this study used a new process of de-novo protein design to computational build a novel protein that would bind to and inhibit the inhibitor. The method was successful in determining a new protein inhibitor of EBV, and so can now we used to design proteins to fight other infectious agents and cancer.
E. Procko, G.Y. Berguig, B.W. Shen, Y. Song, S. Frayo, A.J. Convertine, D. Margineantu, G. Booth, B.E. Correia, Y. Cheng, W.R. Shief, D.M. Hockenbery, O.W. Press, B.L. Stoddard, P.S. Stayton, D. Baker, "A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells," Cell 157, 1644-1656 (2014).
Stimulating Insulin Production in the Fight Against Type-II Diabetes
Treatments for type-2 diabetes have centered on targeting the human receptor protein GPR40, since it is a fatty-acid receptor that can enhance glucose-dependent insulin secretion. TAK-875 is a drug developed by the company Takeda to stimulate insulin secretion by binding to this receptor. Researchers at Takeda have released a publication showing the details of the mechanism of action of this drug for the first time, based on protein structures solved at beamline 5.0.3.
A. Srivastava, J. Yano, Y. Hirozane, G. Kefala, F. Gruswitz, G. Snell, W. Lane, A. Ivetac, K. Aertgeerts, J. Nguyen, A. Jennings, K. Okada, "High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875," Nature 513, 124 (2014).
Keeping the Heart Pumping and the Neurons Firing
Nearly every cell in the human body contains ion channels; conduction of potassium across membranes is what helps keep muscles moving, the heart pumping, and brain neurons firing. And channel proteins also have to be selective: calcium channels selectively drive Ca2+ into cells despite a 70-fold higher extracellular concentration of Na+. These studies revealed the molecular mechanisms by which channel proteins are both selective and efficient.
L. Tang, T.M. G. E.-Din, J. Payandeh, G.Q. Martinez, T.M. Heard, T. Scheuer, N. Zheng, W. Catterall "Structureal basis for Ca2+ selectivity of a voltage-gated calcium channel," Nature 505, 56 (2014).
How Cells Distinguish Between Young and Old
Researchers in this study used crystal structures solved at beamline 5.0.1 to show how the enzyme TAT distinguishes between slow and fast turnover rates of microtubules and marks them accordingly. The work is broad-reaching because marking of microtubules affects cell division, mobility, and lifetime.
A. Szyk, A.M. Deaconescu, J. Spector, B. Goodman, M.L. Valenstein, N.E. Ziolkowska, V. Kormendi, N. Grigorieff , A. Roll-Mecak, "Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase," Cell 157, 1405 (2014).
Understanding the Virus-Antibody Arms Race
Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. This works provides a overview of the structural mechanisms underpinning the virus-antibody arms race.
D. Fera, A.G. Schmidt, B.F. Haynes, F. Gao, H.X. Liao, T.B. Kepler, and S.C. Harrison, "Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains," PNAS USA 111, 10275 (2014).
Cooperative Binding to Brain Receptor Proteins
The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are neuron receptor proteins that signal for muscular contraction upon a chemical stimulus. This study shows for the first time cooperative binding of certain ligands, showing interactions between various domains of this receptor.
K. Kaczanowska, M. Harel, Z. Radić, J.P. Changeux, M.G. Finn, and P. Taylor, "Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein," PNAS, 111 10749 (2014).
Toxins in the Brain
Membrane-embedded receptors in the human brain have myriad responsibilities in human health: pain sensing, learning and memory, nervous system development, and detection of injury. In a series of high-profile studies, these investigators solved the structures of many of these important proteins alone and in complex with toxins, giving critical insight into exactly how these proteins sense and respond to extracellular signals.
L. Chen, K. L. Duerr, E. Gouaux, "X-ray structures of AMPA receptor–cone snail toxin complexes illuminate activation mechanism," Science, 345 1021 (2014).
The Dopamine Receptor Unravelled
This work show for the first time the structure and domain arrangement of the main dopamine receptor in the brain: "...one of the most important receptors in the brain -- a receptor that allows us to learn and remember, and whose dysfunction is involved in a wide range of neurological diseases and conditions, including Alzheimer's, Parkinson's, schizophrenia and depression....". from Nature News & Views: "Neuroscience: A structure to remember."
C.-H. Lee, W. Lu, J.C. Michel, A. Gorhring, J. Du, X. Song, E. Gouaux, "NMDA receptor structures reveal subunit arrangement and pore architecture," Nature 511, 191 (2014).
Stopping a Virus From Keeping Infected Cells Alive
The Epstein-Barr virus not only infects cells, but then blocks cell apoptosis, keeping the infected cells alive so they will go on to spread the infection. In this study, a novel protein inhibitor was designed computationally, then grown, crystallized and structurally determined at beamline 5.0.2, which revealed how the protein binds to the virus and allows apoptosis. The designed protein was then shown to suppress tumor growth and extend survival in animal models.
E. Procko, G.Y. Berguig, B.W. Shen, Y. Song, S. Frayo, A.J. Convertine, D. Margineautu, G. Booth, B.E. Correia, Y. Cheng, W. Schief, D.M. Hockenbery, O.W. Press, B. L. Stoddard, P.S. Stayton, D. Baker, "A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells," Cell 157, 1644 (2014).
Kinase Activity and Cytokine Signalling
Cytokine signaling is essential for cell growth, hematopoiesis, and immune system function. Cytokines can induce dimerization of their receptors, which in turn induces activation of other proteins such as Janus kinases (JAKs) leading to further signalling inside the cell. In this study, researchers used crystal structures obtained at beamline 5.0.1 of certain JAK proteins to deduce a connection between cancer-associated mutations in the protein and unusual kinase activity.
P. J. Lupardus, M. Ultsch, H. Wallweber, P.B. Kohli, A.R. Johnson, C. Eigenbrot, "Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition", PNAS 111, 8025 (2014).
Transporters As Drug Targets
A class of transporters, the ASBT proteins, are under scrutiny as potential drug targets for treatment of hypercholesterolaemia and type 2 diabetes. These proteins are bile acid transporters could also be used for delivering drugs orally, and so their structure is of immense importance to medical research. This study, using structures solved at beamline 8.2.2, revealed some of the first three-dimensional images of these important transporters.
X. Zhou, E.J. Levin, Y. Pan, J.G. McCoy, R. Sharma, B. Kloss, R. Bruni, M. Quick, M. Zhou, “Structural basis of the alternating-access mechanism in a bile acid transporter,” Nature 505, 569 (2014).
The Pain-Sensing Gatekeepers of the Cell
Certain ion channels can detect inflammation or injury by monitoring concentrations of protons outside the cell wall. Using beamline 5.0.2, Gouaux and coworkers solved the structure of one of these ion channels in complex with a pain-inducing toxin from the Texas Coral snake, revealing exactly how this protein responds to extracellular toxins.
I. Baconguis, C.J. Bohlen, A. Goehring, D. Julius, E. Gouaux, “X-Ray Structure of Acid-Sensing Ion Channel 1-Snake Toxin Complex Reveals Open State of a Na-Selective Channel,” Cell 156, 717 (2014).
Architecture of Cell Adhesion
The ability of cells to “stick together” is what makes multi-ceullular life possible, and so cell adhesion molecules have evolved across all major species. The level of “stickiness” is regulated by interactions between cells. Crystal structures solved at beamlines 8.2.1 and 8.2.2, along with biochemical characterization have show the architecture of this adhesion, and have furthermore shown that the proteins involved may do more than just facilitate the interface between cells; they may also be used in intercellular signaling.
E. Ozkan, P.H. Chia, R.R. Wang, N. Goriatcheva, D. Borek, Z. Otwinowski, T. Walz, K. Shen, K.C. Garcia, Cell 156, 482. (2014).
How Plants Deal With Varying Nitrate Levels
Plants require nitrate for proper growth, but the availability of nitrates in soil can vary by orders of magnitude. These studies, using structures obtained at beamline 8.2.1, revealed how a nitrate transporter is able to switch between two states for high or low affinity in order to transport the appropriate amount of nitrate to meet the needs of the plant.
J. Sun, J.R. Bankston, J. Payandeh, T.R. Hunds, W.N. Zagotta, N. Zheng, "Crystal structure of the plant dual-affinity nitrate transporter NRT1.1", Nature 507, 73 (2014).
A Big Step Toward Targeted Gene Control
Bacteria use the CRISPR-Cas9 system to delete foreign DNA that comes from viruses. Now, humans are starting to realize its potential in targeted genome editing and gene regulation. The beauty of the Cas9 system is that the same enzyme can be used to target nearly any gene in the human genome. But how does it work? Doudna and coworkers used crystallographic structures from 8.2.1 and 8.2.2, and cryo-EM structures to determine two major Cas9 enzymes subtypes, and their orientation and binding to DNA.
M. Jinek, F. Jiang, D.W. Taylor, S.H. Sternberg, E. Kaya, E. Ma, C. Anders, M. Hauer, K. Zhou, S. Lin, M. Kaplan, A.T. Iavarone, E. Charpentier, E. Nogales, J.A. Doudna, "Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation," Science, 343 (2014).
Computationally Designing Drugs
This study used computational design to generate small, stable protein that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. The results were validated using structures obtained at beamline 5.0.1. The results not only provide a promising leads for development of virus vaccines for human respiratory illnesses, but also provide proof of principle for a variety of other vaccine targets, such as human immunodeficiency virus and influenza.
B.E. Correia, J.T. Bates, R.J. Loomis, G.Baneyx, C. Carrico, J.G. Jardine, P. Rupert, C. Correnti, O. Kalyuzhniy, V. Vittal, M.J. Connell, E. Stevens, A. Schroeter, M. Chen, S. MacPherson, A.M. Serra, Y. Adachi, M.A. Holmes, Y. Li, R.E. Klevit, B.S. Graham, R.T. Wyatt, D. Baker, R.K. Strong, J.E. Crowe, P.R. Johnson and W.R. Schief, “ Proof of principle for epitope-focused vaccine design,” Nature, 507 201 (2014).
The Versatility of RNA
The replication of RNA without the aid of proteins is thought to have been a critical step in the emergence of simple cellular life from prebiotic chemistry, but the chemical copying of RNA templates generates product strands that contain unusual linkages. However, this research found that such RNAs can still be functional, and that diminished stability is offset by changes that render the global RNA structure relatively unchanged.
J. Sheng, L. Li, A.E. Engelhart, J. Gan, J. Wang, J.W. Szostak, “Structural insights into the effects of 2′-5′ linkages on the RNA duplex,” PNAS, v111(8), 3050 (2014).