Our sense of smell is dependent on recognition of a vast array of odorants despite having a finite number of receptors. Odorants are mostly detected by G protein-coupled receptors called odorant receptors in olfactory sensory neurons. While there are only approximately 400 odorant receptors in humans, combinatorial activation of these odorant receptors enables sensing of odorants with diverse chemical structures. However, the mechanistic basis of odorant binding to odorant receptors in humans has remained unclear.
SBGrid member Aashish Manglik and other researchers have been working to develop a structural understanding of how odorants are recognized by odorant receptors. Using cryo-EM, they report the structure of active human odorant receptor OR51E2 bound to fatty acid propionate.
Above: Structure of human OR51E2 bound to propionate in complex with miniGs399. CC BY SBGrid.
Based on this structure, they determined that propionate is bound within a pocket in the odorant receptor and makes specific contacts to achieve activation. After mutating the odorant-binding pocket, they observed altered recognition of fatty acids with varied chain length. This suggests that activation of odorant receptors by odorants is influenced by tight packing interactions. Through molecular dynamics simulations, they show that propionate induces conformational changes in a specific region of odorant receptor OR51E2, extracellular loop 3. This work provides a foundational understanding of odorant recognition by human odorant receptors at a high-resolution, structural level.
Bacteria can become infected by bacteriophages and have developed a range of anti-phage immune pathways to counteract these infections. These pathways are often multi-gene systems encoding proteins that sense and inhibit virion production, and efforts to catalog anti-phage signaling systems in bacteria have revealed that some of these genes share homology with components of eukaryotic immune systems. This suggests that eukaryotes horizontally acquired some innate immune genes from bacteria.
Many components have been identified as homologous between bacteria and humans, including bacterial cyclic-oligonucleotide-based anti-phage signaling systems (CBASS) with human cGAS and STING, and bacterial Viperins and Gasdermins with human Viperin and Gasdermin D. However, SBGrid member Aaron Whiteley and other researchers have been searching for other potential components in bacterial anti-phage signaling systems which could be homologous to immune signaling elements in humans. The researchers demonstrate that bacteria express anti-phage proteins containing a NACHT module, which is an important element of the animal nucleotide-binding domain leucine-rich repeat containing gene family called NLRs. These NACHT proteins are widespread in bacteria and contain a C-terminal sensor, central NACHT module, and N-terminal effector component, acting against both DNA and RNA bacteriophages.
Above: Previously reported structure of NLR family CARD domain-containing protein 4. CC BY SBGrid.
Importantly, they determined that mutations in human NLR which lead to stimulus-independent activation of downstream signaling also activate bacterial NACHT proteins, suggesting that the bacterial and human systems share similar signaling mechanisms. This work identifies NACHT module-containing proteins as ancient innate immune signaling elements and expands our knowledge of homology between bacterial anti-phage immune pathways and eukaryotic immune systems.
Understanding how iron affects stearoyl-CoA desaturase-1 activity.
Enzymes are the manufacturing facilities of the cell. They take the materials provided by the environment and convert them into cellularly relevant chemicals. The physical processes that allow enzymes to do this vital work are of interest to scientists because of the possibility of treating enzyme-related diseases, using enzymes as therapies for neurodegenerative diseases, and harnessing their manufacturing process for production of chemicals relevant to society. In a recent study by SBGrid member Ming Zhou from Baylor College of Medicine, the maintenance and function of a subclass of enzymes becomes more apparent.
Stearoyl-CoA desaturase-1 (SCD1) is an enzyme that localizes in the endoplasmic reticulum of cells and contains two iron atoms at its core. SCD1 uses the core irons to carry out the catalytic function of adding a double bond to fatty acids, thus creating unsaturated fatty acids. This enzyme contributes to diseases like cancer and diabetes and is a possible target to treat neurodegenerative diseases. Zhou and colleagues pursue an answer to how and why SCD1 loses enzymatic activity after a certain amount of uses.
Above: Published structure of SCD-1 diiron active site. PDB: 6WF2. CC BY SBGRID
Similar to common machines in a household, enzymes stop functioning after a certain number of uses. Understanding why a machine stops working can provide insight about how to keep them working longer and more effectively. Zhou and company used enzyme activity assays to show that SCD1 lost function after 8.5 reactions due to the loss of an iron from the active site of the enzyme. The team used UV-vis spectroscopy, EPR, and inductively coupled plasma mass spectrometry (ICP-MS) to confirm that the loss of activity was due to the loss of iron in the active site. They also found that this iron loss could be remedied by providing an excess of available iron for SCD1 to take up and continue its work, though once SCD1 becomes inactive this treatment can not restore activity. This provides a possible “repair” for low SCD1 activity by ensuring that it has a large pool of possible iron molecules with which to maintain its function. This study provides interesting insight into how to limit and possibly prevent enzyme self-inactivation.
Read more about this work in the Journal of Biological Chemistry.
Understanding the Structural Basis of Broadly Neutralizing Antibody Binding to HIV
More than 35 million individuals are impacted by HIV. There is significant interest in the development of an HIV vaccine, but these efforts have been challenged by glycan shielding of relevant HIV epitopes, low surface density of envelope (Env) spike protein trimers, and viral clade-based and mutation-mediated diversity. Specific broadly neutralizing antibodies (bNAbs) which can bind to HIV viral particles and prevent viral fusion with host cells have been identified and studied in order to improve immunogen design for the advancement of an HIV vaccine. However, these bNAbs demonstrate extensive somatic hypermutations and abnormally long and short complementarity determining region 3 heavy and light chains, respectively.
Many groups have been working to understand interactions between bNAbs and viral Env protein trimers in order to improve immunogen design for an HIV vaccine. Specifically, this work seeks to elucidate the interactions between BG18, a bNAb with the highest known potency against the V3/N332 region of the virus, and HIV Env protein trimers. Since chemical and conformational heterogeneity of glycans on HIV Env challenges crystallization, nearly all solved structures to date have been reported with high mannose-only forms of N-glycans. However, since surface glycans are extensive on these viral particles, corresponding to nearly 50% of the mass of HIV-1, it is increasingly important to understand interactions between BG18 and natively-glycosylated virus.
As a result, SBGrid member Pamela Bjorkman and other researchers have worked to solve a 3.8-Å X-ray free electron laser dataset, of natively-glycosylated Env trimers complexed with BG18.
Above: Structure of BG505 SOSIP.664 HIV-1 Envelope Trimer in complex with BG18 and 35O22 bNAbs. CC BY SBGrid.
Analysis of the molecular footprint of the antibody in this structure suggests that BG18 shares contacts with PGT121/10-1074 bNAbs, but exhibits a different Env trimer-binding orientation which facilitates additional interactions with V3-loop base glycans and proteins of the V1-loop. This knowledge of the structural basis of BG18 binding will enable improved immunogen design to advance the development of an HIV vaccine.
Read more about this exciting work in Nature Communications.
For Bil Clemons, the first glimpse of a protein structure never gets old. “Seeing the electron density for the first time—it’s still just magical and one of the greatest experiences you can have as a scientist,” says Clemons, a biochemist at the California Institute of Technology (Caltech).
Above: The crystal structure of Get4 bound to the N-domain of Get5 with the NMR spectra of the Get5 dimerization domain in the background. Image credit: Courtesy of B.Clemons
Those moments might be all the sweeter because of the months and years of trial and error that culminates in the final atomic image of a protein. As a graduate student, for example, Clemons put in 4 years to help solve the structure of the small ribosomal subunit, part of the molecular machine that translates genetic material into proteins in the cells. Now he works on labor-intensive membrane proteins.
Clemons got hooked on exploring the atomic terrain of molecules in part by a pair of 3D glasses when he was an undergraduate at Virginia Tech. The paper glasses were tucked into a special magazine on structural biology handed out in a class designed to help steer biochemistry students toward potential careers. Clemons ended up at the University of Utah in Salt Lake City. He was the first graduate student to join the lab of newly arrived Venki Ramakrishnan. Why did he make that choice? “I loved the concept of protein translation,” says Clemons.
About four years into Clemons’ PhD, Ramakrishan moved the lab to the MRC Laboratory of Molecular Biology (LMB) in Cambridge, England, largely because of the stable funding for risky projects like the ribosome structure. Clemons made the transition and helped to finish the atomic resolution structure of the 30S subunit. In 2009, Ramakrishnan shared the Nobel Prize in Chemistry for the structure of the ribosome. Notably, Clemons features prominently in Ramakrishnan’s 2018 memoir of the path to the Nobel, “The Gene Machine.”
As a graduate student, Clemons weathered the tedious task of freezing hundreds of crystals one at a time in the cold room by playing music. Clemons, who always has music on in the background, went through a Johnny Cash phase as a graduate student. But truth be told, the saxophone is the family instrument.
Clemons’ father and namesake, William, was a U.S. Marine bandsman and sax player who grew up in a segregated south. His late uncle Clarence was a saxophonist with Bruce Springsteen’s E-Street Band, a gig subsequently inherited by Clemons’ younger brother Jake. Clemons’ mother was an elementary school teacher from North Dakota. Because of the military, the family moved around, but one constant influence on Bil was a passion for understanding the natural world exemplified by the availability of iconic shows such as ‘Mutual of Omaha’s Wild Kingdom’.
When he received his PhD, membrane proteins were becoming a feasible area of structural biology. Contemplating a postdoctoral fellowship, Clemons emailed Tom Rapoport at Harvard Medical School, a leader in membrane protein biogenesis, for advice on what group would be best for working on the structure of the protein translocation channel. Rapoport asked for his CV, then quickly invited Clemons to join his team working on the SecY channel, a team that included Stephen Harrison and members of his group.
At HMS, Clemons and his colleagues determined the structure of the universal channel that allows newly made proteins to be transported through or into cellular membranes. The finding was reported in Natureon New Year’s Day 2004.
“The ribosome was great, because no one had conquered it,” Clemons said. “But we knew what that mountain looked like. The structure didn’t change the general basics of what we knew. But with the protein translocation channel, the structure told us everything. Leveraging the known biochemistry with a structure, we could finally generate mechanistic models that could be clearly tested. It has stood the test of time.”
Membranes create protected spaces for living cells with a bilayer defining an inside and outside. Embedded membrane proteins span this barrier and provide a critical path for information and nutrients. A third of the genome encodes membrane proteins. Most membrane proteins are made next to the membrane, because their hydrophobic sections are incompatible with the watery environment inside the cell. To ensure correct insertion, for most membrane proteins, the ribosome cozies up to the endoplasmic reticulum and pushes the protein through the protein translocation channel, where the protein can be inserted into the membrane. There, it can fold and then, if necessary, be trafficked to various organelles.
At Caltech, Clemons has been studying another pathway used by a class of membrane proteins, known as tail-anchored proteins, to integrate into the membrane. “They have a special toolkit for getting into the lipid bilayer,” he says.
The lab has structurally characterized the various components that take the protein from the ribosome and deliver to the endoplasmic reticulum through the cytoplasm. “It’s a dance, a series of handoffs of the proteins, orchestrated player to player,” he says.
In the process, he has run the gamut of structural biology methods and pretty much all the software packages at SBGrid. But structure is just the starting point for figuring out the mechanism for the proteins in motion. His team probes the dynamics and chemistry using biochemical and biophysical tools “to understand at the deepest level how they’re working,” he says.
In addition to his work on membrane protein biogenesis, the lab has focused on other areas, such as the twin-arginine secretion pathway and membrane associated glycosylation pathways.
The Clemons lab has added another tool: Computational biology. They are trying to take some of the trial and error out of the difficult early stage of studying membrane proteins. “Caltech has enabled me to lean on students with a lot of computational training,” he says. “With that, we’ve tried to think about problems in our area that haven’t been addressed and try to solve them.”
A big problem for structural biologists begins at the first step. Scientists often insert the gene for protein of interest into certain bacteria or yeast, and then induce the host to generate the protein. For membrane proteins, this is a make-or-break first step, because “it is unlikely that one can get enough protein to study,” Clemons says. “Every other step in the process has a high failure rate, but if you don’t succeed in the first step, you will not succeed at all.”
He proposed using computers to design a statistical model that would predict how bacteria will react when asked to create a protein they don’t normally produce. With a key graduate student, his team showed that it is possible to predict membrane protein expression directly from sequence data, which allows researchers to double their odds of expressing a membrane protein. The findings were published in March 2018 in the Journal of Biological Chemistry.
“It’s early days,” Clemons says. “But just to demonstrate that we can leverage computation for this complicated problem is exciting for us. Large data sets, statistical tools and bioinformatics are opening up new paths. The synergy between computation and biochemistry is very powerful.”
Until recently, a vaccinated llama has been a membrane protein crystallographer’s best friend. That was before Andrew Kruse and his co-authors showed that yeast can be a faster, cheaper, and possibly better tool for otherwise impossible crystallographic studies.
Broadly, the Kruse lab at Harvard Medical School is interested in how cells transfer information across their membranes. To probe the finer points of the molecular interactions, Kruse and his collaborators have developed new tools, including the yeast platform and a new evolutionary approach, to make studies of these membrane proteins easier for themselves and others.
Above: This structure of RodA in bacteria was solved using a de novo model constructed using sequence evolution analysis and may pave the way for next-generation antibiotics. Courtesy: A. Kruse.
About 20 years ago, it was discovered that the unique immune systems of camelids (llamas, camels, and alpacas) produce a pared down version of antibodies, featuring a single binding domain called a nanobody. Nanobodies were a boon to structural biologists. Among their many uses, these small molecules stabilize membrane proteins in specific different active conformations for crystallization and detailed structural analysis.
Yet despite the transformative impact on structural studies, it takes months to produce specifically tailored nanobodies by llama immunization. It can be prohibitively expensive, and the particles might not work. As a junior faculty member, Kruse felt constrained by cost and time limitations of immunization. So did a fellow junior colleague, Aashish Manglik at University of California, San Francisco. Together, the labs came up with a solution.
The yeast project arose out of their need for a better way to stabilize G protein-coupled receptors. GPCRs are a proteins lodged in fatty cell membranes that are crucial to human health and disease. The receptors detect hormones and neurotransmitters and then signal other proteins in the cell to respond. Millions of people take drugs targeting GPCRs, such as antihistamines, bronchodilators, antihypertensive agents, and many others. Better understanding of the structure and interactions could help researchers design more effective drugs with fewer side effects.
The yeast platform is one step toward the next stage Kruse envisions for GPCRs: A rational approach to finding and designing drugs to trigger or block specific activity.
“In signal transduction, there’s never been a more exciting time,” he says. GPCRs have both greasy and water-soluble parts, making them difficult to study at the atomic level until recently. The first high-resolution structure of a human GPCR wasn’t solved until the year 2007, Kruse says, but now dozens have been probed in their many different active shapes.
To boost those efforts, Kruse and his collaborators have made the vials of specially engineered yeast and instructions freely available to others for non-profit research. They fielded more than 100 requests from researchers in 17 countries little more than a month after publishing February 12, 2018, online in Nature Structural & Molecular Biology. About half of those requests came from posting the paper on the preprint service, BioRxiv. Other labs are using the nanobodies for projects ranging from identifying inhibitors of the zika virus to detecting bacterial species to classical genetic screens.
For as long as Kruse can remember, he dreamed of becoming a scientist. He grew up in the Twin Cities, Minnesota. Kruse discovered structural biology as an undergraduate at University of Minnesota, as a double major in biochemistry and math. It was a satisfying blend of fundamental quantitative biology with direct applications to human health. He worked in the lab of Douglas Ohlendorf on the structures of bacterial toxins.
Working in the lab also helped open up another part of the world. Because of its growing importance in science, Kruse decided to learn a Chinese language. A lab administrator connected Kruse to a university in northeast China where she had worked for years before settling in Minnesota. Before going to graduate school at Stanford, Kruse spent a summer working at the school in China. There, he met his wife. They speak mandarin exclusively at home. He has no formal collaborations in China, but an acquaintance at the National Center for Protein Science in Shanghai is helping distribute his new nanobody yeast platform within the country.
Kruse began working on GPCRs in graduate school in the Stanford University laboratory of Brian Kobilka. About halfway through Kruse’s PhD training, Kobilka shared the 2012 Nobel Prize in Chemistry with Robert Lefkowitz of Duke University for their discoveries on GPCRs.
In Kobilka’s lab, Kruse worked on a GPCR group called muscarinic acetylcholine receptors. In studies to understand the molecular basis for therapeutic drug action, he showed how tiotropium bromide (Spiriva), an inhaled drug for chronic obstructive pulmonary disease and for uncontrolled asthma, recognizes and blocks the muscarinic 3 (M3) receptor. The once-daily dose is effective in dilating bronchial airways for 24 hours, because the receptor closes around and locks onto the drug, trapping it in place, he found.
He also worked on the related M2 muscarinic receptor, showing how multiple compounds could bind simultaneously to the same receptor. In other projects, he and his Stanford co-authors developed a way to generate more selective affinity reagents for transmembrane proteins. The new method led to a patent and then to a company. In 2016, Kruse was selected by Forbes magazine for its 30-under-30 in health care, along with Aaron Ring, now at Yale University, who together had founded Ab Initio Biotherapeutics, Inc. Ring also collaborated with Kruse and Manglik, another co-founder, on the new yeast platform.
At Harvard, the Kruse lab continues to work on membrane signaling with a variety of approaches, both old and new. For example, they collaborated with another Harvard lab on a mass spectrometry technique to track signaling in living cells.
For a 2017 PNAS paper, they reached back a century to the days when medical researchers used material from nearby slaughterhouses. With a modern twist, they used mail-order frozen calf livers to track down the identity of Sigma 2, a receptor implicated in Alzheimer’s disease, schizophrenia and cancer. Despite its potential therapeutic importance, it had not been cloned. In this case, the lab purified the receptor from homogenized calf livers and revealed its identity as the transmembrane protein (TMEM) 97. They are working on solving the structure. Sigma receptors were discovered in the 1970s because of their response to analogs of drugs that activate opioid receptors. In 2016, the Kruse lab determined the structure of Sigma 1, an unusual receptor that cross reacts with many drugs and has been linked to neurodegenerative diseases, addiction and pain.
In a newer technique, they are using evolutionary analysis and prediction to solve structures from scratch, a collaboration with Harvard systems biologist Debora Marks. Structural biologists need a starting point to determine a new atomic structure, but there was no homologous protein for RodA, a transmembrane protein that bacteria use to build their cell walls. Evolutionary sequence data has been used to predict protein folds, but never to solve a crystal structure of a protein of this size, Kruse says. In this case, they generated hundreds of possible templates and tested them all. A few models were good enough to solve the structure (see image), published online 28 March in Nature. For the computational structure approach, they relied on molecular replacement software implemented by SBGrid, generating a shell script to iteratively run hundreds of searches.
This publication highlight is part of the SBGrid/Meharry Medical College Communities Project, focused on science education and demonstrating how structural biology and preclinical science connect to medicine.
In the body, proteins carry out vital tasks to keep us alive. In order to do the wide variety of tasks needed to keep a living system going, proteins can form unique structures in order to carry out each unique function. These structures are encoded into the protein by its amino acid sequences, which are in turn determined by an mRNA sequence that is encoded by a DNA sequence. Information flows from DNA to RNA to protein structure in the cell and if any problems occur in this information pipeline, detrimental consequences follow. One example of a problem in the pipeline is a mutation in the DNA causing proteins to fold into the wrong shape and not be able to carry out their function. This is the case for Huntington’s Disease. In this neurodegenerative disease, the DNA that makes up the Huntington gene is mutated, almost resembling a skipping record, repeating the same nucleotide phrase, cytosine–adenine–guanine (CAG), over and over again. This repetition causes the resulting protein to have long regions unfolded or misfolded because of repeats of the glutamine amino acid (Gln, Q) and is referred to as a polyQ disease. Proteins with polyQ tracts are enriched in genes with neuronal function and typically aggregate together and cause the tragic symptoms that are hallmarks of the disease. In a recent publication in a IUCr Journal, SBGrid member Dr. Dierk Niessing, publishes a new protein structure that could help limit the aggregation of Huntington gene proteins.
Pictured above is the determined structure of TRMT2A in Drosophila melanogaster. PDB:7PV5. CC BY SBGRID
Recent studies have shown that the inhibition of a protein called TRMT2A can lessen the aggregation of Huntington proteins, which means that TRMT2A could be a potential target for therapeutic molecules. If a drug can inhibit TRMT2A activity, it has the potential to help with Huntington’s disease. In order to determine how well this hypothesis works, we need a model organism that allows for ethical experimentation. Luckily, Dr. Dierk Niessing found a homolog of TRMT2A in the common fruit fly. He used protein crystallography and x-ray diffraction to observe the structure of this homologous protein. Upon examination, this fruit fly homolog had a different sequence than the human TRMT2A protein but a very similar structure. All the catalytic residues, the functionally relevant amino acids in the human protein, were also conserved in the fruit fly protein. This paper builds a case using structural biology that this fruit fly protein is a very similar homolog to the human TRMT2A protein. The implications of these findings mean that fruit flies could be an effective model to study how well drugs that target this protein are able to lessen Huntington symptoms. Read more about this fruit fly homolog in the Acta Crystallographica Section F.
-Vida Storm Robertson, Fisk University
Vida Storm Robertson is a Masters Student in Chemistry at Fisk University working in both solid-state and solution based structural determination techniques. He plans on starting a PhD program in biophysics in the fall of 2024.
This publication highlight is part of the SBGrid/Meharry Medical College Communities Project, focused on science education and demonstrating how structural biology and preclinical science connect to medicine.
Copper is a micronutrient that helps regulate many important functions needed for bacteria to survive. However, high amounts of copper can be toxic to bacteria so it’s important for bacteria to be able to maintain homeostasis. Copper’s main role is to act as a cofactor. A cofactor is a non-protein molecule that is crucial for a protein to carry out its function. Many proteins use copper as a molecular helper; because of this, copper is essential for all living things, yet, it is still unclear how bacteria get and maintain their copper levels. The work titled Stabilization of a Cu-binding site by a highly conserved tryptophan residue by SBGrid member Oriana Fisher looks into how the Gram-positive bacterium Bacillus subtilis regulates copper levels.
Structure of copper(II) (tan spheres) bound to the mutant YcnIW137F. CC BY SBGRID.
The authors looked at the ycnKJI operon that contains three copper-using proteins. An operon is a group of genes that are all controlled by the same promoter, or the place where genes are turned “on”. They found that a member of the ycnKJI operon, YcnI, a copper-binding protein whose function is still unknown, prefers to bind the oxidized version of copper. This oxidized version is known as Copper(II) oxide or Cu(II). By creating a mutated version of Ycnl where they swapped out a conserved tryptophan for phenylalanine, they found that even though tryptophan, an amino acid and binding site of Cu(II), is found in about 98% of this family of copper-binding proteins, it is not essential for Cu(II) binding. The conserved tryptophan, however, is necessary for maintaining the stability of Cu(II) binding to the protein.
Read more in the Journal of Inorganic Biochemistry.
