Peter Kwong was putting the finishing touches on his work at the University of Chicago solving the structure of ?-bungarotoxin, a neurotoxin in snake venom, when structural biologist Wayne A. Hendrickson called from Columbia University. "Would you be interested in working on CD4?" he asked.
The year was 1987. Kwong, who was doing graduate research in the lab of the late Paul B. Sigler, a pioneer in structural biology, was in the process of moving to Yale.
At the time, CD4, the receptor for the human immunodeficiency virus, was so new that Kwong hadn't even heard of it. But he jumped at the opportunity. "That's what's so great about structural biology," says Kwong, who is now chief of the Structural Biology Section at the Vaccine Research Center (VRC) at the National Institutes of Health. "As a graduate student, you can work on the cutting edge."
Kwong, Hendrickson and Seongeon Ryu, another graduate student from the Hendrickson group, published their CD4 solution in Nature in 1990. In 1998, Kwong and Hendrickson solved the gp120 glycoprotein, the part of the HIV virus that allows it to gain entry into host cells and also the part that triggers the production of virus-neutralizing antibodies, in complex with the CD4 receptor and a neutralizing antibody.
By the time Kwong finished graduate school and post-doctoral training at Columbia, about 20 therapies for HIV had been developed, but no vaccine. So he decided to join the fledgling VRC with hopes of contributing to an effective HIV vaccine.
That end has not yet materialized, but Kwong recently turned vaccine development on its head with a new, structure-based approach he calls "antibody to vaccine." Using this approach, he has designed a vaccine for respiratory syncytial virus (RSV), a virus that hospitalizes about 100,000 children in the U.S. each year.
Kwong's approach blends what he calls the "East Coast" and "West Coast" schools of thought on vaccine development: The East Coast school says you need to understand the virus, while the West Coast school says you need to understand the immune system, because it is antibodies from the immune system that do the work.
"I'm somewhere in the mid-West," says Kwong, appropriately so, since he grew up in Chicago. "We start by understanding which antibodies work and then use viral antigens recognized by those antibodies to figure out the immunogens that can recreate those antibodies."
To reverse engineer the natural biological process, Kwong developed an iterative approach, identifying appropriate antibodies and the antigens that elicited them, and then using structural biology to refine the antigens into viable vaccines. The first step involves finding the antibodies humans make that actually work against a virus. Once a human has developed antibodies that effectively neutralize HIV-a, which can take years, it can take years to isolate these antibodies in human sera. So Kwong developed a bioinformatics shortcut called neutralization fingerprinting that allows him to easily pick promising sera samples from infected individuals. Once appropriate samples are identified, then appropriate template antibodies can be identified.
X-ray crystallography drives the next stages of the process, which involve determining the structural features of the antibody and antigen, then engineering that antigen to make it able to elicit antibodies like the original template antibody. "This process allows for a timeline of vaccine development that is potentially much, much faster," says Kwong, who solved the structure of RSV fusion (F) glycoprotein bound to a potent antibody, D25, in November 2012 and had the RSV F vaccine manuscript submitted to Science by July 2013.
SBGrid has been a key enabler for Kwong, whose lab is surrounded by virologists, immunologists, and vaccinologists. "Initially we wondered if we could succeed as an outpost," he says. "But that's what's so wonderful about SBGrid. We can import the latest technology and be part of a cyber community that allows us to be at the forefront of crystallography."
Kwong is now applying these technologies to other viruses, including HIV, though HIV is a formidable target. The variation of HIV in a single individual is equivalent to that of seasonal influenza globally, he says. "It's an enormous challenge."
Published December 17, 2013
Karin Reinisch had being doing structural biology since graduate school, and as a post-doc solved the reovirus core in the lab of Stephen Harrison. But it wasn't until she arrived at Yale in 2001 to set up her own lab that she found her niche. "At least half of the department, six or seven people at the time, were working in one particular area, how you move materials between different organelles," she says. "That made it a very rich environment for a structural biologist."
Even today, Reinisch, now associate professor of cell biology at the Yale School of Medicine, is the only structural biologist in her department (though there are many other structural biologists at Yale). Because Reinisch works with so many collaborators on multiple different aspects of membrane trafficking, her work is "more like thematic collections of short stories than a full-length novel," she says. "Each project is its own distinct entity that will tell you something about a process."
Reinisch began her studies in chemistry as a Harvard undergraduate. Her desire to understand the mechanisms underlying the chemical reactions she was studying led her to pursue structural biology as a Harvard graduate student in the lab of William Lippscomb, winner of the 1976 Nobel Prize in Chemistry.
She worked on the allosteric regulation of enzymes, but the work didn't grab her. "I was more interested in how proteins interact with one another on a larger scale," she says.
So as a post-doc, Reinisch moved to the Harrison lab, where she worked on solving the reovirus core. This protein assembly is very large, approximately 50 megadaltons, an order of magnitude larger than many individual proteins. To work with such a large structure, she had to dig deep into the software to understand what it was doing and to fiddle with it.
In addition, all of the data was collected without freezing crystals. The crystals took nearly a year to grow, an exceedingly long time given that they usually can be grown overnight. In addition, 500 beamline exposures made up the final data set, with at least as many discarded. The results of Reinisch's monumental effort were published in Nature in 2000 and provided novel insights about how double-stranded RNA viruses make and deliver viral messenger RNA into the host cell.
Once at Yale, Reinisch began to investigate the protein complexes involved in various aspects of membrane trafficking. For instance, Reinisch collaborated with her department chair and 2013 Nobel Prize in Medicine winner James Rothman to solve the crystal structure of complexin, a protein involved in the regulation of SNARE proteins at a neuronal synapse. SNARE proteins make up the machinery that allows vesicles to fuse with their membranes and deliver neurotransmitters. "Our model is still controversial, but I'm hoping it will survive the in vivo tests we have for it," says Reinisch.
A major focus of the Reinisch lab is on the proteins involved in membrane identity. There are many different membrane-bound compartments in each cell, each housing different chemical processes and requiring different molecules to be shuttled in and out. "You want to move material to the right place, but how do you know what the right place is?" asks Reinisch. "You need signposts."
Two such signposts are the Rab GTPases and different species of phosphoinositide. She has explored how Rab GTPases are activated and now she is investigating a protein complex that synthesizes phosphatidyl inositol 4-phosphate, a plasma membrane marker. "It's a nice theme," she says, another collection of short stories in the making.
Incidentally, when Reinisch was setting up her lab at Yale in 2001, she had a little help. One of her lab mates in the Harrison lab at Harvard, Piotr Sliz, had just begun streamlining the installation and maintenance of a collection of X-ray crystallography applications in the lab, an effort that marked the very beginnings of SBGrid. He helped Reinisch set up her lab computationally, making her the first member, though at the time unofficial, of SBGrid.
Published November 15, 2013
They say the shoes make the man. For Mishtu Dey, assistant professor of chemistry at the University of Iowa, the shoes made the science.
In 2007, during the last year of her postdoc at the University of Michigan, Dey told her mentor, Steve Ragsdale, that she wanted to crystallize the enzyme she was studying, methyl-coenzyme M reductase (mcr), the only enzyme found in nature that produces methane. Ragsdale, who isn't a crystallographer, gave her the go ahead. "He didn't think I'd actually do it," she says.
The trouble was that oxygen inactivates mcr so that it won't take up the substrates or produce the methane product. "If I don't keep oxygen out of the system, I'll end up with a dead enzyme," says Dey.
So Dey had to figure out how to do anaerobic crystallography on her own in a lab that wasn't even set up for basic crystallography. She set up crystal trays in an oxygen-free glove box and pulled a microscope inside. To see through the eyepiece, she had to tilt the scope towards the plexiglass and look in from the outside. "I had a certain size heel on the shoes I wore to make the height perfect for me to see through," she says.
Heels and all, it was worth it. Day became the first person to crystallize mcr in one of its many redox-active form. "That was really exciting for me," says Dey, who in her lab in Iowa is now trying to crystallize the active form.
Dey had originally trained as a chemist at the Indian Institute of Technology in Mumbai, where in graduate school she worked on supramolecular host-guest chemistry. She had learned crystallography as part of her work designing and synthesizing biomimetic small molecule complexes that could be used for glucose sensing and monitoring in medical devices.
During that time, she found herself asking a pivotal question: Why not work with real enzymes? The question led her to the Ragsdale lab. "Deciding to transition to biochemistry was the biggest decision of my career," she says.
Initially, the switch presented a steep learning curve. "Synthesizing molecules in flasks is a different mindset from growing microbes in a fermenter," she says.
By the time she'd gotten the hang of it, her interest in crystallography had resurfaced, inspiring her to crystallize mcr. Once she had the crystals, she traveled to Chicago from Ann Arbor for days at a time to visit the synchrotron and collect the data.
While exciting, the work was also frustrating because Dey wasn't able to solve the structures on her own. "I decided that if I'm doing so much crystallography, why not go to a crystallography lab and learn the last bit, how to work with the data," she says.
Dey moved to the lab of Catherine Drennan at MIT and worked on another anaerobic enzyme, hydroxypropylphosphonic acid epoxidase (HppE). In 2013, Dey's findings that this antibiotic biosynthetic enzyme's mechanism of action involved an unprecedented reaction were published in Nature.
In 2011, Dey moved to the University of Iowa to start her own lab. "I was looking for a research university with a medical school on campus," she says. She envisioned collaborating with medical researchers and doing work in mammalian systems to study human diseases.
That vision has already materialized. Today, one of the main focuses in Dey's lab is working with human cancer cell lines to study oxygen-sensing metallo-enzymes. These enzymes regulate genetic programs in response to hypoxia, a common feature deep inside tumors. In this work, she combines crystallography with techniques from biochemistry and spectroscopy to understand these enzymes.
The crystallography work at Iowa is easier now that she has introduced the university's protein crystallography core and other crystallography labs to SBGrid. Yet one of her biggest challenges remains the same. "Anaerobic crystallography was really challenging 10 years back, and its the same challenge now," she says. "If you're doing any redox biology or biochemistry, oxygen is always a concern."
Published September 17, 2013
Brian Crane was all set to stay in Canada to attend graduate school when he heard about a new program at the Scripps Research Institute billed as graduate studies "at the interface of chemistry and biology." In 1990, such integrated programs were just emerging, so Crane, a chemist with biochemistry leanings, was intrigued. He had never heard of Scripps, being himself from Manitoba, in Winnipeg, but after a visit to the California campus, he decided the program was a perfect fit.
"The emphasis was chemical, but it had a biology culture," he says. "More freeform and discovery based."
At Scripps, Crane studied metalloenzymes and applied structural biology techniques to trying to understand the catalytic mechanisms of two types of redox enzymes, sulfite reductases and nitric oxide synthases. He focused on trying to characterize the chemical kinetics of the catalytic reactions, such as understanding the timing of protein and electron transfers between cofactors.
Crane continued this work after graduate school in a fellowship at California Institute of Technology, where he combined his background in crystallography with spectroscopic methods and also learned how to use photochemical techniques to initiate electron transfer reactions. He extended this work to begin looking at electron transfer across protein-protein interfaces. "Electron transfer had always been difficult to study in solution," he says. "But in crystals, you can lock everything down and use different tricks to get the electrons to move."
Later, in his own lab at Cornell, where he is now a professor of chemistry and chemical biology, Crane got interested in the broader biological context in which these enzymes operate. He decided to investigate how photochemistry and electron transfer reactions influence two biological sensory systems: light-sensitive circadian clocks and redox-sensitive bacterial chemotaxis.
"What I like about the circadian clocks and chemotaxis systems is the protein networks are tied intimately to the behavior of the organisms," says Crane. "Small changes in these proteins, as much as the mutation of a single residue, can greatly alter behavior, so it gives you a way to go directly from biochemical reactivity to behavior, which is quite amazing."
As an example, in Crane's studies of fungal light sensors, he has developed modified versions of the light-sensing proteins that have different chemical kinetics in response to light and dark cycles. "When we introduce these modifications into the fungi, we see pronounced changes in fungal behavior in terms of circadian rhythms," he says.
To study the circadian clock in a biological context, Crane collaborates with experts in fly and fungal biology. In his own lab, the biggest challenges involve working with the unstable protein complexes that govern these clocks and the large membrane assemblies that provide bacteria with sensory input. "The whole is greater than the sum of its parts," he says. "Figuring out how these proteins work together is a daunting task. You can't quite imagine how they really work by just looking at the pieces."
For instance, in bacterial chemotaxis, the assumption was that the receptors outside the organism talked to downstream effectors in a linear system. But recently Crane's lab defined an elaborate two-dimensional hexagonal lattice inside the membrane that provides the interface. "It works as a huge allosteric enzyme that allows one receptor to activate 35 kinases on the other side," he says. "You can't understand how this works at all unless you grasp the complexity and build up these larger structural models."
To do this work, Crane is integrating X-ray crystallography with electron microscopy and pulsed dipolar electron spin resonance spectroscopy. "It quickly becomes a computational problem," he says. "Often we want to try something new, so we go into SBGrid. Sure enough, there'll be something there that we can use to get started."
Published August 16, 2013
Mark Lemmon's career in structural biology began with a decision not to pursue structural biology. At Oxford University as an undergraduate, he'd been drawn to understanding biochemistry at a structural level. But the thing he wanted to understand most, transmembrane signaling, posed a problem. "In the late 1980s, solving crystal structures of membrane proteins wasn't something one could expect to do," says Lemmon, George W. Raiziss Professor of Biochemistry and Biophysics at the University of Pennsylvania.
So he decided to study membrane signaling using molecular instead of structural techniques. His graduate work at Yale University involved making lots of mutations in the transmembrane region of the epidermal growth factor receptor (EGFR) and collecting data on which mutations affected dimerization.
At that time researchers had only just begun to understand the 58 tyrosine kinases in the human proteome, of which EGFR is one. "In the beginning, everyone assumed they all worked the same way," says Lemmon. "But later we realized that they all have their foibles."
After completing his PhD, Lemmon did a post-doc in a pharmacology lab at New York University, where he shifted his focus from the part of the protein that sits within the membrane, just 2% of the whole EGF receptor, to the intra- and extracellular domains.
He also shifted his methods. "I basically joined a non-crystallographer's lab and started getting more interested in crystallography," says Lemmon.
Lemmon's goal was to understand cellular signaling and molecular regulation, but he wanted to understand this at the structural level. While at NYU he collaborated with the late Paul Sigler, a prominent structural biologist at Yale, and Kate Ferguson, then a student in Sigler's lab, and now Lemmon's wife.
In parallel, as basic curiosity drove Lemmon and others to understand the mechanisms of these important signaling molecules, clinical researchers had begun to recognize that kinases with mutations often become oncoproteins.
Today Lemmon's lab focuses on the clinical relevance of these kinases. "Now more than ever we need to understand the details of how these receptors work so that when a mutation appears in the clinic, we can know what it means from our knowledge of mechanism and structure," he says.
Lemmon is collaborating with oncologists at Penn who have sequenced the ALK gene in nearly 2000 neuroblastoma tumors and found 50 different mutations. About 30% of those mutations are not active cancer-drivers, so ALK-inhibiting drugs will likely have no effect.
The problem is, no one knows which mutations are active and which are silent. So Lemmon is looking at each mutation structurally, biochemically and even pharmacologically to understand which mutations matter. "We're bringing structural biology in to personalized medicine," he says.
Lemmon is also about to launch a similar project focusing on EGFR. "I'm going back full circle to where I started," he says, trying to solve the structure of this large transmembrane protein and understand how the full-length receptor really works. The challenges are still "enormous," he says, so he is employing multiple methods in addition to crystallography, including mass spectrometry and electron microscopy.
Lemmon, as chair of his department, wants to lower the bar for researchers to use all the methods they need, so he and six other labs at Penn have signed on to SBGrid. "Suddenly labs that have an inkling that they want to do crystallography, who've been scared of it before, now have everything in place," says Lemmon. "Their biggest challenge is getting the data rather than analyzing it."
Published July 16, 2013
A brush with fame leaves Jane Tao longing for the lab and her studies of the proteins that coat viruses
A brush with fame leaves Jane Tao longing for the lab and her studies of the proteins that coat viruses
In 2006, Yizhi Jane Tao accepted an award for being one of the most influential Chinese at her undergraduate alma mater, Peking University. Other awardees included Oscar-winning director Ang Lee and actress Zhang Ziyi, who starred in Crouching Tiger, Hidden Dragon.
"It was good to see that our work is appreciated," says Tao, associate professor in biochemistry and cell biology at Rice University. "But it made me realize that fame is not important to me. I like my work better."
Tao studies viruses, specifically RNA viruses. She got her start working with bacteriophages in graduate school at Purdue University in the lab of Michael Rossmann, one of the first labs to solve a 3-D viral structure. After graduate school, she trained with Stephen Harrison at Harvard Medical School, another pioneer in solving virus structures.
Viruses are not only different from other organisms, but also from one another. "Viruses are small but very clever," says Tao. "They can adapt to many hosts and environments."
Her first major discovery, in 2006, identified a loop in the nucleoprotein of the H1N1 human influenza virus. Every negative-sense RNA virus encodes a nucleoprotein that surrounds the virus genome and allows it to replicate. In this case, Tao found that the protein has a distinctive loop that is essential for self-oligomerization and encapsidation of the viral genome into a double-helical hairpin structure.
The road to that discovery was fraught with trial and error, both in Tao's labs and those of others who had tried to solve this structure. The protein was hard to nail down because it tends to form different sized oligomers. "It's not homogeneous," she says.
Finally her lab found a strain of the virus that produced mostly trimers that could be crystallized and solved. With the protein structure in hand, Tao identified the loop and its function. She then compared the amino acid sequences of the nucleoprotein from different influenza viruses, finding that the oligomerization loop is a well-conserved feature.
Since then, small molecules that block this loop have been found to block infection in cell culture, such as nucleozin, reported by a group at the University of Hong Kong. A group led by Chi-Huey Wong at National Taiwan University is continuing the work, finding nucleozin analogs and also finding more precise targets within the nucleoprotein. That drug development is ongoing, but Tao remains focused on her work solving the structures of virus proteins.
Tao later solved the structure for the capsid protein coat of a fungal double-stranded RNA virus. The stunning image, published in 2009 and pinpointing the locations of 5 million atoms, was created with a combination of data from x-ray crystallography and electron microscopy. "It looks very beautiful," says Tao, who is one of SBGrid's earliest members, having joined in 2002. She used the RAVE package, developed by Gerard Kleywegt's group in Uppsala, Sweden and distributed by SBGrid, to solve this structure.
The capsid of this relatively small double-stranded RNA virus is distinct from many other bigger double stranded RNA viruses, such as the rotavirus and reovirus. "They probably evolved on a different pathway from the bigger ones," she says.
Tao's lab focuses today on trying to understand how the viruses package the genome, and how the capsid changes its structure to inject the viral genome into a cell. While much of Tao's work is medically relevant, particularly her work on the influenza virus, after all these years of work on these viruses, that aspect of the work is less important to her than it was originally. "Mainly, it's just interesting," she says. "I want to see how it works."
Published June 14, 2013
While the bacterial toxin that causes anthrax has been used as a deadly biological weapon, from a scientific point of view, it has an upside. "The nice thing about anthrax is that separate proteins make up the toxin," says Borden Lacy. "So long as you keep them apart, it's entirely safe."
If there are nice things to say about other bacterial toxins, she will likely know them. They are her specialty.
Lacy, an associate professor of Microbiology at Vanderbilt University, began her studies of toxins in graduate school at the University of California, Berkeley by looking at the botulinum neurotoxin, which causes muscle paralysis and death at extremely low doses. Of all the toxins Lacy has worked on, it is "the most worrisome," she says. "We had to get vaccinated and we had a special room for working on it."
When Lacy began working on this bacterial toxin and potential biological weapon, no one knew what it looked like. In the late 1990s, she determined the structure and found that the protein looked very different from other bacterial toxins.
The toxin assumes one shape when it makes its way into an endosome in a cell. But as the endosome evolves, its pH drops, causing the toxin to change from a soluble form to a membrane-inserted form, which allows it to form a pore in the membrane. "It was exciting to be the first person to get a glimpse into how this might occur," she says. "It's also guided our thinking about how we could inhibit the toxin or use it for other purposes."
Later, as a post-doctoral fellow in John Collier's microbiology lab at Harvard Medical School, Lacy wanted to know how the anthrax toxin invades cells. Because the toxin proteins can be studied separately, Lacy didn't have to work directly with the anthrax bacterium. Instead, she used E. coli to produce sufficient quantities of the individual proteins to form crystals for x-ray crystallography studies.
Lacy isn't always this fortunate. Recently, in a study of the botulinum toxin progenitor complex, she had to forego x-ray crystallography and instead use electron microscopy because regulations prohibit her from having more than half a milligram of the substance. "Sometimes we just can't have enough material to really pursue getting crystals," she says.
In 2006, Lacy moved to Vanderbilt University, one of SBGrid's biggest sites, along with her husband, Ben Spiller, also a structural biologist and former post-doc at HMS in the lab of Stephen Harrison. They both joined the Center for Structural Biology, which centralizes equipment and resources. "The center makes it easy to use multiple techniques," she says. "It's very open and collaborative and has a critical connection with SBGrid."
As a result, Lacy has applied crystallography, nuclear magnetic resonance, electron microscopy and other methods to her projects and uses several SBGrid-installed applications to do so, including HKL2000, Phenix, Coot and others.
Today, Lacy's work is focused on Clostridium difficile, a bacterium that plagues hospitals. Patients become vulnerable to the bug when they take antibiotics that disturb the normal gut flora. At its mildest, C. diff can cause diarrhea, but at its worst, it can cause life-threatening complications and has a high rate of recurrence.
The bug produces two large protein toxins, weighing in at approximately 300 kilodaltons, so Lacy is collaborating with Melanie Ohi, a former HMS post-doc from the lab of Thomas Walz, to study their structures by electron microscopy. She is also breaking the toxin into smaller domains to study with x-ray crystallography, understand their mechanisms, and potentially discover therapeutic targets.
"In this case maybe more so than some of the other toxins I've worked on, I can clearly see how understanding these structures could help us identify the most effective neutralizing antibodies or small molecule inhibitors or even inform vaccine design," says Lacy.
Published May 21, 2013
Pushing the Boundaries of Technology
In the mid-1960s when Stephen Harrison began to determine the structure of the tomato bushy stunt virus, SBGrid didn't exist. There was no need for it. They didn't even have a hard disk for storage.
"Near the end of the 60s they got a disk. That was a big deal," recalls Harrison, Giovanni Armenise-Harvard Professor of Basic Medical Sciences at Harvard Medical School. "One disk."
Lacking storage and a network, as a doctoral student in biophysics at Harvard, Harrison had to walk to the Computer Center to code his programs on punch cards. The limiting factor, however, wasn't so much the burden of manual labor as the cost of CPU time. The billing algorithms for the mainframes scaled with a program's use of random access memory (RAM), which by the early '70's still maxed out at a megabyte.
"There was a big premium on writing programs very efficiently and over-writing locations the instant you no longer needed the information," says Harrison, who worked with punch cards and mainframe technology through to 1977, when he completed the final computations of the structures of the bushy stunt virus at better than 3 angstroms resolution.
"One could not have done it more than a year or two earlier," says Harrison. "We pushed forward more or less as fast as the computing resources allowed."
Laboratory-based DEC and VAX computers debuted in the 70s, accelerating the pace of science. "We could do our day-to-day computations in the laboratory, and we could do them over and over again and make mistakes without pouring money down the drain," says Harrison.
But the DEC computers that ran film scanners were still so RAM constrained that they could not hold a scan of the film used to collect the X-ray data, which, at the time were recorded using conventional laboratory X-ray generators. Harrison's programs did piecemeal scans, doubling back when necessary.
Later, in the 1980s and into the 1990s, it became feasible to record crystallographic data at shared synchrotrons, and X-ray detection transitioned from film to electronics and, more recently, direct pixel array detectors, such as the PILATUS collector used at the Northeastern Collaborative Access Team (NE-CAT) facility at APS. NE-CAT is a shared synchrotron dedicated to X-ray crystallography at the Argonne National Laboratory, managed by Cornell University, and shared by researchers, including Harrison, at multiple institutions in the northeastern US.
Today, says Harrison, biological problems in X-ray crystallography are, for the most part, no longer computationally limited. As a result of the recombinant DNA revolution, neither are they biologically limited by the need to find a purifiable protein or complex of interest in large quantities in nature.
Rather, says Harrison, "they are limited by whether or not we can find an embodiment of an interesting biological problem in a molecule that crystallizes."
As a result, some of Harrison's current projects, related to human virus structures, are moving away from X-ray crystallography toward electron cryomicroscopy (EM). EM technologies are advancing rapidly and, with the recent introduction of direct electron detectors, reaching a new level of maturity. "I think over the next five years the boundary will move toward electrons and away from X-rays for problems that have no electron solution today," says Harrison.
As an example, Harrison is trying to use EM to investigate interactions of human antibodies with the influenza hemagglutinin glycoprotein. As an immune response develops, antibodies mutate toward higher affinity. Understanding such antibody affinity maturation is a key part of vaccine development.
If EM proves to be a more straightforward and rapid way to solve these structures, Harrison speculates that EM may make its way into the vaccine development process much the same way X-ray crystallography has become an essential tool for drug development.
Meanwhile, EM image analysis is pushing the boundaries of available computing, says Harrison. He is, once again, moving forward as fast as the computing resources allow.
- Elizabeth Dougherty
Published April 22, 2013
A full plate keeps young investigator Joseph Ho relaxed, creative, productive
In graduate school at Boston University, Meng-Chiao (Joseph) Ho nearly quit science. He had chosen to focus on a difficult problem, solving a perfectly twinned protein crystal without a homology model to use for phases. His mentor, Karen Allen, a biochemist and crystallographer, had already spent 12 years trying to solve the structure. After 5 more years and no results worth reporting, Ho had a choice: graduate without a paper to his name or give up.
"I almost became a chef," says Ho, who is now an assistant research fellow at the Institute of Biological Chemistry, Academia Sinica in Taiwan. "What's the difference between a chef and a biochemist? I already knew how to cook by recipe."
After a trip home to Taiwan to renew his visa, Ho gave the project one more shot by working on orthologs to find an untwinned candidate. This time he and Allen succeeded.
Ho published the findings in Nature and landed himself a post-doctoral fellowship in the laboratory of Vern Schramm, chair of the Department of Biochemistry at the Albert Einstein College of Medicine. An expert in drug discovery, Schramm maintained a steady state of over a dozen projects, with ten postdocs, at least one being a protein crystallographer.
When Ho arrived in 2007, he filled this role, even though so far he had only solved three structures. By the time he left, he had worked on at least fifty structures and had published ten papers. "This was a fantastic time for me to learn the ins and outs of solving structures," says Ho, who also worked under structural biologist Steven Almo, a longtime collaborator with Schramm.
The crush of simultaneous projects suited him. "When you have multiple projects, you always feel comfortable," says Ho. There's always enough work to allow for breaks from the tough problems and progress on the straightforward ones.
With the pressure off, says Ho, he "accidentally" solved another difficult problem: a ricin toxin bound to one of Schramm's inhibitors. In one of many attempts to form a crystal with the ligand inside the active site, Ho took too long to fish out his crystals so the crystal dried out. Since he had some beam time available at Brookhaven National Laboratory, he collected the data anyway. "It wasn't a clear picture, but there was something in the pocket and the crystal lattice had shrunk a bit," he says. Using a "poor-man's dehydrator" (cutting the well open to allow crystals dry out and waiting), he eventually hit just the right level of dehydration and solved the structure.
In 2011, when Ho moved back to Taiwan to build his own lab, he took these two experiences as key lessons. "No one should focus on just one crystallography project," he says. "The chance of a blackout is too high." To support his multi-project idea, he built his own medium throughput protein production platform.
Ho also decided to join SBGrid. Having been involved in software upgrades at Albert Einstein, he knew the challenges of keeping the systems up to date and synchronized. "With SBGrid, everything is upgraded at once and our work stays consistent," he says.
In keeping with his interest in drug discovery, Ho chose to zoom in on a pharmaceutically important family of enzymes, protein arginine methyltransferases (PRMTs), which regulate many cellular functions, from gene regulation to cell growth. PRMTs also interact with multiple protein substrates, so there are many projects to work on.
Ho and collaborators recently solved the Xenopus form of a PRMT5-MEP50 complex, important in embryonic development and aberrant in some forms of cancer. Ho reported the findings within months of publication of the human structure by scientists at Eli Lilly and is now investigating the possibility that the complex may be relevant for substrate selectivity.
- Elizabeth Dougherty
Published March 18, 2013
A look inside the Utah-based lab of Wesley Sundquist
Wes Sundquist got his first taste of structural biology as a doctoral student in chemistry at MIT in Cambridge, MA, designing small molecules to bind to DNA and using nuclear magnetic resonance imaging and crystallography to look at them.
"The more I looked at the molecular biology, the more the biomolecules interested me," says Sundquist, professor of biochemistry at the University of Utah. When Sundquist completed his degree in 1988, he swapped one Cambridge for another, spending the following 4 years as a post-doctoral fellow at the Medical Research Council Laboratory of Microbiology at Cambridge University, UK.
While there, he worked under Aaron Klug, winner of the Nobel Prize in 1982 for crystallographic electron microscopy and for solving the structures of nucleic acid complexes. "They were tackling what seemed to be important problems," says Sundquist, who while there studied telomeric DNA structures, the hairpin-shaped tails that cap chromosomes.
In 1992, Sundquist moved to the University of Utah and started his own structural biology lab. The biochemistry department at the university had just received a grant from the Markey Foundation to launch a structural biology initiative. "It was clear the group was going to grow," says Sundquist.
Today, Sundquist is one of the earliest members of a large, multifaceted and collaborative department, making it possible for him to use NMR, EM, Xray crystallography, genetics and biochemistry in his work. The still-growing department has a strong NMR program with an NMR Center at the Medical School, as well as experts in electron microscopy (Adam Frost) and protein crystallography (Chris Hill).
Over time the group has had several influential members, including Tom Alber, now at the University of California, Berkeley, and Venki Ramakrishnan, now at MRC LMB, who won the Nobal Prize for his work describing the structure and function of the ribosome. He solved the first ribosome structure at Utah.
SBGrid membership helps the Sundquist lab find and use the right tools and methods for their work. The lab joined the SBGrid consortium just a year ago. Since then, "we're a lot more adventurous in trying to do new things," says Steve Alam, research assistant professor, NMR spectroscopist, and SBGrid liaison in the Sundquist lab.
Sundquist's work today focuses on the HIV virus. When he started his lab, many people were studying the enzymes in the virus, but few were looking at the structural proteins. So Sundquist filled the space. "We thought the structural proteins might help organize the different steps in the viral lifecycle," he says. "I think that's turned out to be true."
Most recently, Sundquist has been working out the mechanics of how HIV uses host pathways to replicate and, specifically, to bud from cells. The virus hijacks a pathway, ESCRT, used by the cell to complete the final step in cell division. Sundquist found one of the two known ways in which the virus links into this pathway. "We believe we've found a third way, so we're working on that now," says Sundquist, who hopes his findings will point to potential new targets for HIV interventions.
Sundquist is also looking forward to working with a new expert in his department, Janet Iwasa, formerly from Harvard Medical School. Iwasa uses scientific animations to test structural hypotheses, such as those Sundquist has about the steps involved in the HIV lifecycle. "I think she's going to change the way we think about these steps and how we visualize them," he says.
Published February 12, 2013
Emil Pai trained as a classical chemist in the mid-1970s at the University of Heidelberg
Emil Pai trained as a classical chemist in the mid-1970s at the University of Heidelberg. He spent his time learning messy, inefficiently named chemical reactions. "A 60 percent yield was cause for celebration," he says.
Then he attended a lecture on enzyme catalysis, a way to perform very precise biochemical reactions. "For a chemist, it was a humbling experience," says Pai. "I realized that to understand these reactions, you have to know what the molecules look like." Pai, now a professor of biochemistry, medical biophysics and molecular genetics at the University of Toronto (and also former PhD advisor of SBGrid director, Piotr Sliz), has been solving structures ever since.
At the Max Planck Institute for Medical Research, Pai earned his PhD, learned X-ray crystallography from Georg Schulz, and learned to make protein crystals from Heiner Schirmer. He also sat near Wolfgang Kabsch, often acting as a guinea pig for his XDS software. "Even today, I know my XDS," he says.
Pai and Kabsch were part of a group that collaborated to solve the first actin structure, a challenge because muscle proteins polymerize and don't form crystals. When a colleague observed that mixing actin with DNase stabilizes the protein, the group applied the technique to get a crystal. It took another 15 years to solve the structure.
In 1991, Pai moved to the University of Toronto to help build up a structural biology community there. Today there are 10 other structural biology labs spread across Toronto, all members of the SBGrid Consortium, with several investigators using NMR and electron microscopy to determine protein structures.
In Toronto, Pai applies crystallography to gain a more complete understanding of an HIV neutralizing antibody that may help in the design of an HIV vaccine.
He is also working to understand a magnesium channel membrane protein, corA. In late 2012, he published a paper in the Proceedings of the National Academy of Science describing a partially opened channel. Though magnesium is correlated with disease, the channel isn't a target for any medical applications yet. "You have to be cautious with magnesium," says Pai. "It's all over the place. If you remove it, you don't have a targeted change."
While still in Heidelberg, Pai had participated in applying a technique called time-resolved crystallography to p21, an oncoprotein, making it possible to watch the structure change over time, as in a video. In the past few years, he renewed his interest in this work. "We hit on a system that we thought would be perfect for time-resolved crystallography," he says.
The system is a defluorinase, an enzyme that breaks carbon-fluorine bonds, the strongest bond in organic chemistry and a barrier to cleaning up environmental pollution. So far, Pai has found that the enzyme and its complexes crystallize and diffract at high resolutions, he can make a lot of it, and he is also able to run the chemical reaction that moves the compound's parts within the crystal without destroying it.
The only thing missing is a good trigger to initiate the reaction. "Sometimes the chemistry does you in," says Pai. He is hoping to find an organic chemist up to the challenge of solving the problem.
- Elizabeth Dougherty
Published January 11, 2013
Structural Biologist Axel Brunger leaves no method unturned
Axel Brunger joined SBGrid in the early days, in 2006, but he may be best known among structural biologists as the man behind CNS (the Crystallography & NMR System), which he contributes to SBGrid, among other tools. Today, however, Brunger focuses almost all of his work on understanding the molecular mechanism that causes neurons to release neurotransmitters and propagate nerve signals.
"Most drugs for treating neurological diseases affect postsynaptic signaling," says Brunger. "If we could develop compounds that could act on the actual release, that could open up a range of more specific and finely tuned drugs."
Long before exploring neuronal proteins, Brunger made his mark in computational biophysics. In 1987, he released a program called X-PLOR for the derivation of three-dimensional structures using NMR data. This program became the basis for crystallographic refinement by simulated annealing by implementing restraints for X-ray diffraction data.
Later, he and his collaborators developed CNS, a "second generation" system. "We wanted to expand the scope to include all aspects of crystallographic phasing and to create a self-contained system," says Brunger. CNS includes Brunger's refinement methods, such as cross-validation and simulated annealing, as well as density modification methods and methods to obtain phase information by MAD phasing or molecular replacement.
In addition to speeding structure determination and improving validation of crystal structures, CNS also ushered in a new approach to crystallographic programming. "Instead of writing one opaque program that the user can't see into, we decided to develop a scripting language," says Brunger. He and his colleagues at Yale University, where he developed most of CNS, coded their algorithms and methods in scripts and used a FORTRAN program to do the heavy computational lifting.
Most recently, Brunger, now at Stanford University, together with Gunnar Schröder, now at the Forschungszentrum Jülich, and Michael Levitt at Stanford, developed a new refinement method, called deformable elastic network (DEN) refinement, and implemented it in CNS. In collaboration with Piotr Sliz at Harvard Medical School, this computationally intensive DEN-refinement method has been made available through the SBGrid Science Portal. "You prepare and test the refinement script file locally, then slip it onto the server. It will submit as many jobs as needed to get the best possible solutions," says Brunger, who with Sliz describes the work in January 2012 issue of Biological Crystallography.
In parallel, in the mid-1990s, Brunger decided to pursue experimental science. "At that time, people had just discovered some of the key proteins involved in neurotransmission, such as the SNARE family of proteins," he says. "But there were no structures for any of them." Having done some work on membrane protein structure prediction, it seemed like a good system to explore. In the late 1990s, he and Reinhard Jahn, now at the Max Planck Institute of Biophysical Chemistry, determined the structures of some of the key proteins and complexes involved in the process of synaptic vesicle fusion.
In 2000, Brunger moved his lab to Stanford University because of its close proximity to the SLAC National Accelerator Laboratory and the establishment of an interdisciplinary initiative called Bio-X. His many recent experimental accomplishments include determining the first structure of a botulinum neurotoxin in complex with a SNARE protein and the elucidation of how botulinum neurotoxin recognizes its target on the neuron.
Brunger also employs single-molecule fluorescence microscopy methods to complement his structural studies. "Crystallography only gets you snapshots," he says. "So we use optical microscopy to get at the dynamics and the molecular mechanism."
While today, almost 100 percent of Brunger's lab focuses on experimental biology, Brunger expects a resurrection of computational work in his lab inspired by the first hard X-ray free-electron laser at SLAC. "It will allow us to look at very small crystals or those that are extremely radiation sensitive," he says. "But there are a lot of challenges around how to optimally analyze and interpret such data."
Published October 1, 2012
Applying the data analysis methods of structural biology to understand biological systems
When Zbyszek Otwinowski, who joined SBGrid in the Spring of 2012 along with 8 other laboratories at the University of Texas Southwestern, came from his native Poland to the United states 31 years ago, structural biology was not on his radar. He had come to the University of Chicago to study physics.
But just two years later, he met the late Paul Sigler, a pioneer in crystallography, who worked on the structure of RNA and regulatory complexes. After that, Otwinowski's path shifted away from physics and towards biology. Otwinowski, now a professor of biochemistry at UT Southwestern Medical Center, joined Sigler's lab in Chicago and focused on methods. "I covered it all, from building X-ray equipment and optics to developing computational methods," he says.
This work led him to develop several software products for structural biology. Specifically, he collaborates with Wladek Minor, professor of molecular biology at the University of Virginia, to develop HKL2000 and HKL3000. These software packages are collections of tools that analyze X-ray diffraction data and touch on many stages of the structure solution pipeline.
One of the primary focuses in this line of Otwinowski's work today involves trying to handle the uncertainty inherent in the crystals themselves. When solving structures, one simplifying assumption is that all of the crystal samples have exactly the same structure. But crystals of the same protein vary, radiation damages them, and even a single crystal may contain domains with distinct structural variations, exposed differentially when the crystal is rotated during data collection. "These non-isomorphisms are quite challenging in terms of data analysis," he says. "If the object we are measuring is changing, it creates serious problems in structure determination."
In some cases, this variability isn't critical. "It is more likely to be encountered in frontier-of-science projects," says Otwinowski, who recently collaborated on two such cases, providing his expertise to create custom structure solutions. One was the very large WAVE protein complex, published in Nature in 2010. Another was a chromatin complex of Co-REST and LSD1, published in 2006. He is now working to implement the data analysis advances prompted by such projects, such as estimates of non-decay radiation-induced changes, in an upcoming version of HKL2000.
Otwinowski's work on the chromatin complex, which is involved in histone demethylation, one of the critical steps in the control of gene transcription, triggered an interest in a broader question of chromatin organization. After years of study, Otwinowski and colleagues realized that there are additional, not yet discussed levels of chromatin organization.
Specifically, he saw that the various data analysis methods used to solve structures could be applied to analyzing sequencing and gene expression data to understand cells' transcriptional regulation in new and unexpected ways. "This work touches on systems biology," he says. "There's probably much more to the story of the chromatin structure."
Otwinowski only recently joined SBGrid, though he has been using the tools distributed through SBGrid for decades. "We hope SBGrid will simplify our jobs," he says.
Published August 22, 2012
As a child growing up near Sandia National Laboratory in New Mexico, surrounded by physicists and chemists, Anna Pyle had an unconventional sort of chemistry set. Among her playthings was a cube of depleted uranium (only "slightly radioactive," she says). With the language of science as much a part of her life as English, Pyle, now William Edward Gilbert Professor of Molecular, Cellular and Developmental Biology and Professor of Chemistry at Yale University, chose to study chemistry as an undergraduate.
It wasn't until graduate school at Columbia University that biology grabbed her attention. She began by studying how small molecules recognize DNA. Eventually, however, the mysteries of RNA lured her in. "I realized that RNA was so much more complex and the we didn't understand the design principles, so I set out to attack that," says Pyle.
As a post-doc, she joined the lab of Thomas Cech, who won a Nobel Prize for
his insights into RNA self-splicing, a process that involves segments of
RNA that snip themselves out and then stitch the remains back together.
Once on her own, Pyle zoomed in on group II introns, a type of
self-splicing RNA that can mobilize to different sites on DNA. To get a
handle on the structure of these ribozymes, Pyle synthesized them in the
lab, iteratively tweaking them and observing biochemical changes.
Then, in 2008, her lab solved the first X-ray crystal structure of a group
II intron. Out of necessity during that process, Pyle developed a novel
mathematical approach to represent the RNA backbone that both reduces
complexity and allows fundamental features of the structure's architecture
to shine through.
Recently, Pyle used this mathematical approach to create libraries of discrete RNA conformations that can be used to build RNA structures using electron density data and, in the future, data from cryoelectron microscopy. The work was published in the Journal of Molecular Biology in March, 2012. "Our approaches are going to be useful for predicting structures and even modeling de novo, which we're working on now," she says.
In other experimental work, Pyle recently solved the crystal structure of a complex of double-stranded RNA and RIG-I, an intracellular receptor that recognizes viral RNAs and triggers an anti-viral response. She also studies viral RNA replication in hepatitis C virus. "Most of what we work on has some kind of relevance to infectious disease," says Pyle, who hopes this line of work might lead to better drugs for treatment of hepatitis C and other viruses.
As an SBGrid member, Pyle is both user and contributor of the software tools developed in her lab, such as RCrane for building RNA structures in Coot. "SBGrid is just an amazingly convenient and helpful service," she says. "But perhaps more importantly, it is also a community that enables researchers to share ideas and resources."
Since Pyle began to study RNA in 1990, the field has cracked open. "We realize now that RNAs, especially long, complicated RNAs, are extraordinarily abundant, yet we only a have a handful of crystal structures that show us the molecular details," she says. "We really have a lot of work to do."
Published July 18, 2012
Structure-Guided Protein Design for a Better HIV Antibody
For several years now, the lab of structural biologist Pamela Bjorkman, Max Delbrück Professor of Biology at California Institute of Technology, has been trying to find a new way to stop HIV with antibodies that prevent the virus from infecting a cell.
Normally, the body forms antibodies on its own. But in the case of HIV, which mutates rapidly and has few handholds for antibodies to grab onto, scientists have had trouble uncovering natural antibodies. Only recently have the numbers of natural neutralizing HIV antibodies been significant enough to enable researchers to compare them with one another. Bjorkman's work starts there. She uses structural biology to look at these antibodies and then she uses what she’s learned from structures to design better ones.
In a recent study, published in Science in December 2011, Bjorkman's team used X-ray crystallography to look at how a set of natural antibodies bind an HIV spike protein called gp120. The antibodies selected had shown high potency and breadth and also were between 85 and 96 percent identical in sequence. This similarity allowed the researchers to tease out which unique features of the structures make each antibody work well. "Ron Diskin, the first author, is very good at this," says Bjorkman. "He's just staring at these structures and figuring out what should be where."
Using this method, Bjorkman and her colleagues have designed a new antibody that introduces a single amino acid change to a natural HIV antibody. The changed antibody maintained its breadth and improved its potency an average of 10-fold. "When we compared the original to our mutant, there wasn't a single HIV strain that the mutant was worse against," says Bjorkman. "It was always better."
At worst, the mutant was 2 times better. At best, 2000. Such increased potency means the designer antibody could be used therapeutically at much lower concentrations. "That's an enormous practical difference in terms of whether this is realistic for therapies or prophylaxis," says Bjorkman, who is now working to include this antibody in clinical trials. In addition, she has another unpublished designer antibody with two amino acid changes that is even better than her first.
At CalTech, Bjorkman' work is supported by a facility called the "Molecular Observatory," which hosts crystallization robots and helps support the CalTech beamline at the Stanford Synchrotron Radiation Lightsource. In addition, she relies on SBGrid, something that she didn’t have back when she worked as a graduate student at Harvard Medical School, training with the late Don Wiley. "Nothing was automated then," she laughs. "We grad students spent most of our time writing conversion codes to get files from one program to another."
One data gathering technique Bjorkman is experimenting with (using SBGrid software including ATSAS) is small angle X-ray scattering, in which the protein is in solution rather than in a crystal. "You can't get an atomic resolution 3D structure," she says. "But you can learn things about shape and radius of gyration and behavior in solution."
Story: Elizabeth Dougherty Image: Micheline Pelletier / Gamma
Published April 25, 2012
Viral proteins as promising new targets for cancer therapies
Marc Kvansakul decided to become a structural biologist as a young teen after watching a documentary that described proteins as assemblies of Lego-like blocks. Today Kvansakul’s newly formed lab in the department of biochemistry at La Trobe University in Victoria, Australia, is using what he has learned about the sequences and structures of anti-apoptotic viral proteins to start developing new treatments for Burkitt lymphoma, a form of the disease known to be caused by the Epstein-Barr virus.
Not exactly the same as building yellow and red plastic-brick scale models, but, says Kvansakul, “it’s all very good fun.”
Kvansakul began this line of research during post-doctoral work under structural biologist Peter Colman at the Walter & Eliza Hall Institute in Melbourne. First they studied M11L, a Myxoma pox virus protein that helps viruses survive by disabling an infected host cell’s self-destruction program. Kvansakul and Colman wanted to understand how.
The challenge was particularly interesting because M11L, amino acid sequence-wise, looks unlike anything else. But Kvansakul found that, crystal structure-wise, M11L looks a lot like a known mammalian anti-apoptotic protein called Bcl-2. “We uncovered a completely new area in sequence space that forms the same fold,” says Kvansakul, who used SHARP and PHASER to solve the structure, Coot to build the model and CNS to refine it. He published the findings in 2007 in Molecular Cell.
Now, he says, “we’re going fishing” for other viral proteins that, sequence-wise, look a little like M11L to see if they also turn out to be anti-apoptotic. So far, in his own lab at La Trobe, he has found a few hits suggesting there may be a conserved sequence pattern that builds this important fold. “The fact that we found a molecule that forms the same fold but is only 3% identical on a sequence level tells us that there surely must be loads of these proteins in lots of viruses,” says Kvansakul. Only they can’t be found with a computational search. “Once we work out the biology, we can think about how we can build a search algorithm to find other proteins,” he says.
After that 2007 study, Kvansakul and Colman completed a study of BHRF1, a viral protein in Epstein-Barr virus that blocks apoptosis. Kvansakul found that the viral protein blocks multiple pro-apoptotic proteins in host cells, and that in a mouse model of Burkitt lymphoma it also renders chemotherapy agents ineffective, making it a potent oncogene. The results, also obtained using SHELX, PHASER, Coot and REFMAC, appear in 2010 in PLoS Pathology.
Now in his lab at La Trobe, Kvansakul is working with a medicinal chemist to identify small molecules that block BHRF1’s anti-apoptotic function. “Out of me just being interested in structures has turned into, ‘Oh, why don’t we develop new treatments for Burkitt lymphoma?” he says.
While setting up his lab in 2010 and worrying about how he would manage he lab’s computing needs, Kvansakul happened upon mention of SBGrid online. “I thought, this is sent by Heaven!” he says.
Being in Australia, Kvansakul didn’t make it to SBGrid’s June 2011 Symposium personally. Instead, he invited his lab over for a late dinner and then, around 11pm, they all settled into his lounge and watched the live web feed until 7am. “During prime party hours the students were happy to listen to the speakers and take notes,” he says. “It was brilliant.”
As a graduate student and post-doc, Kvansakul used O—“praising and cursing Alwyn Jones in equal amounts”—and CNS and a few other tools. “The more I work, the more tools I learn about,” and happier he is that that he has SBGrid to manage it all.
As an example, at a conference this summer, he heard about a new technique recently implemented in CNS called deformable elastic network refinement (DEN). “I got home and a couple of weeks later, there was an upgrade from SBGrid and we could run it,” he said. “This is exactly why I signed up with these guys. There are probably lots of things out there I haven’t heard of yet, and hopefully I’ll start running into them too.” —Elizabeth Dougherty
Published December 13, 2011
Finding new ways to help patients with cystic fibrosis
Most people, armed with tartar-control toothpaste and a miniature scrub-brush, do battle with biofilms every morning. Biofilms form when bacteria attach to a surface, like a tooth, and form a colony encased in a protective coating.
SBGrid member Lynne Howell, senior scientist at The Hospital for Sick Children (SickKids) and a biochemistry professor at the University of Toronto, studies biofilms formed by Pseudomona aeruginosa, a bacterium that afflicts Cystic Fibrosis (CF) patients by forming biofilms inside their lungs. She recently uncovered the structure of AlgK, an outer-membrane lipoprotein on P. aeruginosa that helps the bacteria form a biofilm.
For CF patients, the genetic mutation that causes the disease also causes a chloride imbalance in the lungs. The altered lung environment mutates the bacteria and encourages a change from the typical, free-floating form, in which they are susceptible to antibiotics, into colonies encased inside a biofilm that protects them from antibiotics.
These biofilms not only shield the bacteria from drugs, they also help them evade the human immune system. “A lot of the damage to the lungs in cystic fibrosis patients is caused by the immune response that is trying, but failing, to get rid of the bacteria,” says Howell.
Recently Howell's lab, with the help of SBGrid programs Phenix and CCP4, among others, solved the structure of AlgK, a protein required for P. aeruginosa to produce alginate, the polysaccharide that forms the bacterial biofilm inside the lung. Her work, published in the Feb 10 2010 Structure, shows that AlgK helps situate the porin AlgE on the outer-membrane pore, allowing it to export alginate outside of the cell.
Howell's lab has followed up this work by solving the structure of the export porin AlgE, which was published in the July 21 2011 issue of the Proceedings of the National Academy of Sciences. Together the structures of AlgK and AlgE suggest a novel mechanism for polysaccharide export across the outer membrane. By understanding the structures, and chemical and physical properties of the proteins that export the polysaccharides that form the biofilms, she hopes to uncover new ways to battle these lethal biofilm infections.
In parallel work, Howell is investigating an organelle, the type IV pilus, that anchors the bacterium surface in the first place. “If we can understand how this organelle is assembled, we may be able to prevent it from assembling and allowing surface attachment,” says Howell. Without surface attachment, the bacteria lose their virulence.
Regardless, the end game remains the same. “We're trying to improve the lives of kids with this disease,” says Howell.
– Elizabeth Dougherty
Published May 17, 2011
Ning Zheng's drug discovery ideas come from human pathology and plant biology and ...
When Ning Zheng got side-tracked from his studies of protein degradation, he never expected to end up in the plant world. Today, Zheng, associate professor of pharmacology at the University of Washington and an HHMI investigator, runs a triplicate of research agendas, all rooted in Xray Crystallography, and all aiming to find new therapeutic drugs for human diseases.
Zheng started his career solving large protein-protein complexes of ubiquitin ligases and the proteins they bind with to degrade them. Malfunctions in this process of ubiquitination are involved in several diseases including cancers, neurological disorders and viral infections. “A lot of companies are trying to find small molecules to manipulate ubiquitin ligases, but nobody has succeeded,” says Zheng.
In 2005, Zheng got side-tracked. A group in Indiana discovered that a plant hormone called auxin, which regulates plant growth, binds directly with ubiquitin ligase to regulate protein degradation. One year later, Zheng solved the structure of this complex.
By doing so, he discovered a surprising phenomenon. He found that the plant ubiquitin ligase and its target protein have an imperfect interface. Auxin, says Zheng, acts like “molecular glue” by filling in the gaps in this weak binding and allowing the proteins to bind with high affinity.
Zheng is now trying to translate this finding to human pathology, where mutations in human ubiquitin ligases cause low affinity bindings to target proteins. “The idea of using a small molecule, like auxin, to rescue a protein-protein interaction is new in the pharmaceutical industry,” says Zheng.
Once Zheng added the study of small molecules to his repertoire, his research took yet another side road. “All of the sudden science led us to study surface membrane proteins, including hormone receptors and ion channels,” says Zheng. “Most drug targets are on the membrane.”
Zheng, being a membrane protein rookie, had what he calls a stroke of beginners luck when he and his team solved the structure for a voltage-gated sodium channel, a channel others in the field had long been working to solve. “We took a new approach, with crazy ideas,” says Zheng. “SBGrid also helped us. Their software is now available to us and we used it for analyzing these membrane proteins.”
This work, published in the journal Nature, may help drive the discovery of new drugs such as local anesthetics and antiarrhythmics, which directly target this ion channel. Meanwhile, Zheng will let science be his guide.
– Elizabeth Dougherty
Published May 13, 2011