Two recent papers from the Joshua-Tor lab at the Cold Spring Harbor Laboratory highlighted proteins that play an important role in control of microRNA. MicroRNA are short RNA fragments used to "silence" or turn-off certain genes, thus regulating cell proliferation. Deregulation of microRNAs in cells has been linked to numerous cancers, and so understanding how they are controlled (and how they get out of control) is important.
C.R. Faehnle, J. Walleshauser, L. Joshua-Tor, "Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis," Nat. Struct. Mol. Biol. 24, 8:658-665 (2017). doi: 10.1038/nsmb.3428. Epub 2017 Jul 3.
E. Elkayam, C.R. Faehnle, M. Morales, J. Sun, H. Li, L. Joshua-Tor, "Multivalent recruitment of human argonaute by GW182," Molecular Cell 67, 1-13, August 17, 2017.
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)