Research Reports , a feature service by the Office of Research Reports of the National Institute of General Medical Sciences, is designed to let you know what some of our grantees are doing. A list of some recent NIGMS grant awards is also included.
Special Structural Biology Issue
Why Structure? The Structure of Photolyase: Using Light to Repair DNA Designer Drugs May Close in on AIDS A Step Closer to Understanding How Proteins Fold Still a Long Road
This issue focuses on recent research in structural biology that promises to increase our understanding of, or ability to treat, medical problems.
This issue of Research Reports focuses on structural biology, a field in which researchers determine and study the detailed, three-dimensional structure of biological molecules. But why do scientists care about molecular structure? After all, it's the function of a molecule that counts, isn't it?
Molecular structure is important to scientists because it is often related to function, just as household tools are shaped to perform specific tasks. The long neck of a screwdriver enables one to use it to pry open lids, or to tighten screws in holes. The depressions in an egg carton are designed to cradle the eggs so they won't break. A funnel's wide brim and narrow neck enable the transfer of liquids into a container with a small opening.
Structural biologists, who are interested in learning which structural elements are involved in a molecule's function, usually focus on proteins, the type of molecule that does most of the work in biological systems. Most proteins are somewhat spherical, a shape that gives little clue to their function. Some, however, are dead give-aways--collagen is a long, thin protein that provides strength to our skin and joints; DNA polymerase III is a ring-shaped enzyme that slides easily around the DNA it replicates; and hexokinase, which metabolizes sugar in the body, has an open "mouth" that closes down on the sugar it binds.
In addition to enhancing their general knowledge about biological molecules, researchers hope that structural studies may actually help them design new molecules for use in medicine, research, and industry. If scientists know which critical structural features are important for the function of a given protein, they might be able to synthesize the smallest functional unit of that protein for commercial use. Such an application may grow out of the work of Dr. Aziz Sancar and coworkers, who are studying a protein called photolyase that may one day be used in gene therapy for those prone to skin cancer.
Structural biology also provides insight into the biochemical mechanism by which a protein normally operates. Eventually, such knowledge may enable scientists to control the function of a protein in living systems. This would be particularly valuable if the protein is involved in causing disease, or if it is useful in biotechnology or industry. Such a protein--HIV protease--is the subject of study by Dr. Paul Ortiz de Montellano and coworkers, who are designing novel drug therapies to combat AIDS .
Although detailed, three-dimensional structures are extremely useful, determining them is very labor-intensive. Some structures require several years to work out. In the meantime, scientists usually have access to the amino acid sequence of the protein. For decades, researchers have attempted to use a protein's sequence to accurately predict its three-dimensional structure. Recently, Dr. George Rose and his postdoctoral fellow Rajgopal Srinivasan developed a computer program called LINUS that uses amino acid sequences to produce some of the most accurate estimates to date of three-dimensional protein structure.
Although these researchers are using different techniques, studying different molecules, and have different goals, they are united by their desire to better understand biological molecules through structural studies. This enhanced understanding may eventually provide us with such everyday benefits as new pharmaceuticals, materials, and industrial chemicals.
This year, more than 30,000 Americans will develop melanoma--the most malignant form of skin cancer--and 6,000 will die, according to estimates from NIH's National Cancer Institute. But if humans possessed a single enzyme found in many organisms, some of those deaths might be prevented. The enzyme, called photolyase, repairs damage to DNA caused by the sun's ultraviolet rays--damage that could result in skin cancer.
Photolyase, which is present in organisms as diverse as the bacterium E. coli , rattlesnakes, and kangaroos, catalyzes an unusual chemical reaction. The enzyme uses the sun's visible light to reverse the harm done by shorter-wavelength ultraviolet rays.
The sun's ultraviolet light causes damage to DNA by fusing together two of DNA's building blocks--specifically, two molecules of thymidine. The resulting double thymidine (thymidine dimer) forms a bulge in DNA. This bulge usually prevents DNA from being copied, which is necessary for cell division, and from being transcribed into RNA, which is necessary for the DNA to be used to synthesize proteins. Cells thus crippled eventually die, and their damaged DNA is rendered harmless. But when a cell manages to copy its DNA past a dimer bulge, a mutation almost always occurs at the site of the dimer. The resulting daughter cells survive, and pass on their potentially carcinogenic mutations to new generations of cells.
Human cells protect themselves from these mutations using a complex, 16-protein process called excision repair, in which a stretch of DNA containing the thymidine dimer is laboriously cut out of the DNA and replaced by an entirely new stretch. In many other organisms, however, photolyase alone deftly splits apart the thymidine dimer while it is still in the DNA.
The study of photolyase has occupied the entire 22-year research career of NIGMS grantee Aziz Sancar, M.D.,Ph.D., a professor of biochemistry and biophysics at the University of North Carolina School of Medicine, Chapel Hill. Dr. Sancar has received support for this research from NIGMS for the last 14 years. During that time, he and his coworkers have used a wide variety of laboratory techniques to reveal much about photolyase's mechanism of action, and its sequence similarities with other proteins.
"We did all the biochemistry and biophysics that one could do to understand how photolyase works," Dr. Sancar said. "But those methods can only take you so far."
The next step, Dr. Sancar said, was to determine the three-dimensional structure of the enzyme. To do that, Dr. Sancar collaborated with x-ray crystallographer and Nobel laureate Dr. Johann Deisenhofer of the University of Texas Southwestern Medical Center at Dallas. Their three-dimensional crystal structure of the enzyme was published this summer in Science .
Now, instead of estimates and assumptions, Dr. Sancar's group has at its fingertips the actual orientation of and distance between all the atoms in the molecule. Not only did this help Dr. Sancar and his coworkers refine and better understand data from their earlier work, but it also revealed characteristics of the enzyme that would be impossible to determine any other way.
For example, computer models of the three-dimensional structure revealed that photolyase is shaped like South America with a small hole in the center. This hole is the perfect size to hold a DNA thymidine dimer.
This finding suggested an obvious mechanism for photolyase's action. When photolyase binds to DNA, the thymidine dimer appears to stretch at a precarious angle out of the DNA and into the hole in photolyase, where it is repaired.
Despite all the earlier work characterizing photolyase, this "pull-out" mechanism was a surprise. "We never imagined that the enzyme would bind to DNA, pull out a dimer from within DNA, and put it into a hole in the enzyme. There's no way of finding that out but by x-ray crystallography," Dr. Sancar said.
Although the mechanism was unexpected, it is not unprecedented. Recently, three other crystal structures of enzymes that modify or repair DNA have revealed a similar pull-out scheme. Together, these structures suggest that this mechanism might be used by many DNA-binding proteins, particularly those involved in DNA repair. If that is the case--which can only be confirmed by future crystal structures--then the photolyase structure will not only teach scientists about the mechanism that photolyase uses to repair DNA, but it will provide a better understanding of this whole class of proteins and binding interactions.
And scientists aren't the only ones who have taken notice of the work of Dr. Sancar's group. Within a month of publication of the structure of photolyase, Dr. Sancar was contacted by a major chemical company that is interested in using photolyase in sunscreen lotion. A skin-care company is supporting a German colleague of Dr. Sancar's to do more biophysical work on photolyase for the same reason.
Although industry interest is a bit premature--getting photolyase into the nucleus of cells where it can repair DNA would be a significant challenge--Dr. Sancar believes that such an application might be possible in the next decade.
The most likely application of research on photolyase would be gene therapy for those with a rare disease called xeroderma pigmentosum. Those with the disease have a defect in their excision repair system and develop multiple skin cancers in childhood. They rarely live beyond their 20's. If photolyase were correctly inserted into their cells, it could repair at least some of the DNA damage that might otherwise result in cancer.
The crystal structure of photolyase provides scientists with a possible route toward such future applications. With the structure, scientists can evaluate which parts of the enzyme are necessary for its function and which parts are expendable. Organic chemists might then be able to synthesize a molecule containing the smallest functional unit of the enzyme. This synthetic molecule, which would be smaller and more manageable than the entire enzyme, might also be more readily introduced into human cells. There are several organic chemistry labs already attempting to synthesize such a molecule.
Dr. Sancar's group's next project is to determine the structure of photolyase bound to a strand of damaged DNA. Because the enzyme is activated by visible light, the researchers plan to touch off the reaction using a laser. They will then use x rays from a synchrotron to observe the reaction as it occurs. With the resulting data, Dr. Sancar and his colleagues will be able to produce a "movie" of the enzyme in action. They will then be able to study, frame-by-frame and in three dimensions, how photolyase repairs DNA.
This new synchrotron technology is just now being applied to enzymes that act on small molecules, and it has never been used to study enzymes that act on macromolecules such as DNA. If successful, this synchrotron movie will not only provide unparalleled insight into the action of photolyase, but will also open up a whole new technology for studying large biomolecules.
Hearst J. The structure of photolyase: using photon energy for DNA repair. Science 1995; 268:1858-66.
Park H-W, Kim S-T, Sancar A, Deisenhofer J. Crystal structure of DNA photolyase from Escherichia coli . Science 1995; 268:1866-72.
Lights in the auditorium dimmed as the speaker displayed a colorful slide of the 3-D structure of HIV protease, an enzyme crucial to the AIDS virus. The enzyme's structure was decorated with balls of color like a Christmas tree. Each colored ball indicated a structural variant of the enzyme that had successfully evaded an anti-AIDS drug. It was a compelling visual reminder to the scientists present of how fast their target is moving.
The seminar was presented at the 1995 AIDS Structural Biology meeting sponsored by NIGMS. Scientists from around the country gathered to discuss strategies to design drugs against one of their most elusive targets--the AIDS virus.
A common structural strategy for combating the virus has been to attack one of its enzymes. Most current drug therapies, including AZT, target the viral enzyme reverse transcriptase, which is essential for HIV to incorporate its genetic material into a cell. Because the AIDS virus eventually becomes resistant to all these therapies, scientists turned their attention to another viral enzyme, HIV protease, which is required for the virus to replicate. As the colored slide made clear, this approach is also susceptible to the virus' uncanny ability to vary the structure of its enzymes.
Yet there has been some success--in December, the FDA approved the first anti-AIDS drug therapy that targets HIV protease. The drug, designed to be used in combination with AZT, is extremely expensive, difficult to synthesize, poorly absorbed, and includes a warning about possible cross-resistance with future protease inhibitors. But it is considered a major advance in anti-AIDS therapy. At least two other protease inhibitors are in clinical trials.
Researchers continue to search for a single, highly effective protease inhibitor with minimal side effects. One such researcher is NIGMS grantee Paul Ortiz de Montellano, Ph.D., a professor in the Department of Pharmaceutical Chemistry at the University of California, San Francisco.
Dr. Ortiz de Montellano and his collaborators have focused their attention on two amino acids in the center of HIV protease. Also found in related enzymes, these amino acids--two aspartate residues--are necessary for HIV protease to bind to its substrate and catalyze the virus' relentless spread in the body. No variants of HIV protease have been isolated in which these amino acids were mutated. And for good reason: previous studies have shown that when these amino acids are altered, HIV protease and all related enzymes are inactivated. If Dr. Ortiz de Montellano and his coworkers can design a drug compound that targets these critical aspartates, they might have HIV protease--and hence the AIDS virus--by the throat.
In the past, most structurally based drug design involving HIV protease has focused on blocking the interaction of the enzyme with its substrate. Typically, scientists have attempted to design a drug that temporarily latches onto HIV protease and physically blocks binding of the enzyme's natural substrate. But the approach is not as effective as researchers would like, and it fails in the presence of some structural variants of HIV protease.
So Dr. Ortiz de Montellano and his colleagues are attacking the problem from a different angle. Instead of temporarily blocking HIV protease from interacting with its substrate, they are developing special molecules to bind tightly and permanently to the enzyme.
The researchers are starting with the conventional blocking compounds, to which they are attaching reactive chemical groups that, upon contact with HIV protease, fuse with the critical aspartate residues and permanently inactivate the enzyme.
This approach is more difficult than the temporary blocking approach, because it requires the scientists not only to find a compound that binds to the aspartate residues, but also to find a reactive group that inactivates the enzyme, to attach the two, and then to ensure that the end product does not react with any other molecules in the body. Finally, before a potential new drug can be sent to the front lines, it must be fine-tuned to bind to HIV protease much better than does the enzyme's own substrate. The result would be a highly specific, highly effective protease inhibitor.
Dr. Ortiz de Montellano's group is off to a good start. They have two compounds that bind to the aspartate residues, and an arsenal of about 10 reactive groups with which to arm these compounds. That gives them 20 raw recruits to pummel into shape.
If the work proves successful, AIDS meetings in the future might replace the ironically festive, color-coded HIV protease structure with a new structure showing the permanent inactivation of the enzyme by one of Dr. Ortiz de Montellano's molecules. And a battle will have been won in the war against AIDS.
For 40 years, scientists have tried to determine how proteins fold into the three-dimensional shapes crucial for their function. Now they're one step closer.
Proteins control nearly every system in our bodies. Antibodies, enzymes, and many hormones are proteins. A protein's three-dimensional structure determines which molecules it binds, which reactions it catalyzes, and, to some extent, where it is located in the cell.
It is much easier to determine the order, or sequence, of amino acids in a protein than it is to determine the protein's three-dimensional structure. Although the sequence contains all the information necessary for correct folding, scientists still cannot predict a protein's final shape from its sequence.
"If we could decipher the structures of proteins from their sequences, we could better understand all sorts of biological phenomena, from carcinogenesis to AIDS," said James Cassatt, Ph.D., director of the NIGMS Division of Cell Biology and Biophysics. "Then we might be able to do more about these disorders."
Now, NIGMS grantee George Rose, Ph.D. and his postdoctoral fellow Rajgopal Srinivasan at The Johns Hopkins University in Baltimore, Maryland, have developed a computer program called LINUS that promises to come closer than ever before to cracking the protein folding code.
"There are a lot of programs to predict secondary structure--small-scale, localized twists in the protein--from sequence information," said Dr. Cassatt. "But somehow Dr. Rose gets the overall structure. That makes his program unique."
LINUS is named after the late Nobel laureate Dr. Linus Pauling (or, as Dr. Rose said, the name is "99 percent Pauling and 1 percent Peanuts," referring to the popular comic strip). The computer program is based on very simple assumptions. No two atoms are allowed to be in the same space at the same time; amino acids are encouraged to be in the conformations most commonly seen in proteins; and hydrophobic, or "water-fearing," amino acids are encouraged to cluster in the protected center of the protein.
In its most blatant simplification, LINUS doesn't even use the true structure for amino acids in the protein. Instead, it makes a gross approximation. It replaces the side chain--the unique part--of every amino acid with a single sphere whose size depends on the type of amino acid. Normal side chains range in size from 1 to 18 atoms and occur in a variety of shapes, including ring structures, straight chains, and branched chains.
After being fed the sequence of amino acids in a protein, LINUS begins to predict the protein's structure by dividing the sequence into overlapping, bite-sized chunks. Starting with chunks only six amino acids long, LINUS randomly twists the amino acids into any of four possible localized secondary structures: helix, sheet, turn, or coil.
The process repeats 5,000 times, with each trial conformation ranked according to how energetically favorable it is--a measure of how likely it is to occur in nature. If a conformation is ranked as favorable in more than 70 percent of the trial structures, and it enables interaction between some amino acids, it is frozen in position for subsequent cycles.
LINUS then starts another cycle by biting off larger overlapping chunks--12 amino acids this time--and twisting them into 5,000 new conformations. As before, LINUS freezes the most favorable conformations from this cycle, creating a new starting point for the next cycle. The process continues, with LINUS taking ever-larger bites of the protein each round. The final structure is made up of the conformations that are most favored in the last cycle.
The resulting structures provide a surprisingly good estimate of the protein's three-dimensional shape. X-ray crystallography can provide more structural detail, but it requires months, even years, to complete a crystal structure.
"Right now LINUS gives the same benefit as a poor crystal structure," Dr. Rose said. "What is that useful for? A lot of things." For example, he said, it could be used in conjunction with the Human Genome Project to classify proteins that might be involved in genetic disorders. Or it could be used to speed the progress of targeted drug design.
"The most surprising things about this work is how these exceedingly simple ideas, crudely expressed, can do so well in giving you the gross conformation of the protein," Dr. Rose said. Of the seven structures that Dr. Rose examined in his first paper about LINUS, the program predicted the gross overall shape for all but one. Even more impressive, the predicted length and location of the more localized secondary structures--the helices and sheets--were almost identical to known structures determined by x-ray crystallography. For example, examining the sequence of a protein called fatty-acid binding protein, LINUS assigned the incorrect secondary structure to only 14 of the protein's 131 amino acids. The protein has a good number of secondary structures: 2 helices, 11 sheets, and 6 turns.
With further refinements, Dr. Rose expects the accuracy of the program to increase dramatically. For example, in his first published work on LINUS, Dr. Rose told the program to take bites no larger than 50 amino acids. Now he is modifying the program to produce accurate structure predictions using much larger chunks. He also plans to enable LINUS to predict special protein features such as disulfide bonds, prosthetic groups, and multiple subunits. Eventually, he hopes that LINUS will be useful to researchers all over the world.
"Our goal is to boil this program down into something that is sufficiently simple and sufficiently exportable that we can give it to many other labs," Dr. Rose said. "That way, instead of trying to do all these experiments ourselves, other people who are experts in certain areas can try their own experiments."
The process that LINUS uses--identifying small, localized structures, then incorporating them into the overall protein structure--is called hierarchical condensation. Although this technique has been successful in predicting protein structure, Dr. Rose does not claim that it predicts the pathway by which proteins fold in living systems.
"We do think that hierarchical condensation is what happens in one way or another when proteins fold," Dr. Rose said. "But I don't think LINUS is giving a detailed representation of any folding pathway."
Although the field of protein folding is very popular now, that wasn't always the case, Dr. Rose said. "In 1979 when I got my first NIGMS grant to work on this, protein folding was considered a problem too hard to be solved in this century," Dr. Rose said. "Now we're very optimistic."
Srinivasan R, Rose GD. LINUS--A hierarchic procedure to predict the fold of a protein. Proteins: Structure, Function, and Genetics 1995; 22(2):81-99.
Structural biology may help stimulate a revolution in the pharmaceutical industry. By enabling researchers to design, on a molecular level, drugs to bind to specific target molecules, it may spur the development of pharmaceuticals that are more effective and have fewer side effects. But those drugs must still go through rigorous clinical trials, typically the most time-consuming step in getting a new drug to pharmacy shelves.
Traditionally, scientists identify new drugs either by fiddling with the structure of existing drugs or by screening thousands of chemicals and plant extracts from around the world to see if they are active against cell cultures of disease-causing agents. Frustrated chemists often have to analyze 10,000 compounds before finding one that shows potential.
Knowing the structure of a molecular target speeds the identification of promising compounds. It also enables scientists to make more educated guesses about how to modify a potential drug compound to improve its potency or decrease its side effects. After they have identified a candidate compound, researchers must determine whether the compound binds correctly under the appropriate conditions, and, if it does, how to get it into the right place in living human cells, how to prevent it from being metabolized before it reaches its target, and how to ensure that it doesn't produce toxic side effects.
For the health and safety of patients, many aspects of drug development will remain the same. But a slow revolution has begun. Already, structural studies have aided the development of more than a dozen drugs that are now in clinical trials. The technology has also played a major role in the development of a new class of anti-AIDS drugs, the HIV protease inhibitors. Most major drug companies have devoted a department to the pursuit of structurally based drug design, and several smaller companies have been established for this express purpose. There may even be a time in the future when drugs developed using the principles of structural biology will outnumber conventionally discovered ones.
This sampling of titles from the more than 4,000 grant awards NIGMS makes each year is designed to give you an idea of the basic research the Institute supports. A computer printout of all current (new and continuing) NIGMS grants at your institution is available from us, if you request it. Among the awards that were made in the first half of 1995 are the following:
Boston College Charles S. Hoffman: " S. pombe cAMP Signal Pathway"
Brandeis University Christopher Miller: "Basic Mechanisms of Ion Channel Function"
Case Western Reserve University Philip P. Garner: "Asymmetric Synthesis of Bioactive Alkaloids"
Colorado State University James R. Bamburg: "Structure and Function of Actin Depolymerizing Factor"
Cornell University, Ithaca Valley J. Stewart: "Genetic Control of Nitrate Respiration in E. coli "
Emory University Gordon G. Churchward: "Mechanism of Conjugative Transposition"
Florida State University John G. Dorsey: "Amino Acid/Peptide Transfer Thermodynamics"
Johns Hopkins University Floyd R. Bryant: "Enzymatic Transformation of the DNA Helix"
Massachusetts General Hospital Jeevendra J. Martyn: "Alterations in Neuromuscular Function Following Burns"
Massachusetts Institute of Technology Linda G. Cima: "Novel Material for Liver Cell Culture and Transplant"
North Carolina State University, Raleigh Bruce S. Weir: "Statistical and Quantitative Genetics"
Oregon Health Sciences University Michael P. Kavanaugh: "Basic Amino Acid Transporters"
Pennsylvania State University-University Park Jerry L. Workman: "Mechanisms of Transcriptional Regulation in Chromatin"
Princeton University Lee M. Silver: "Control of Genomic Imprinting by a Mouse Imprintor Locus"
Rockefeller University Titia de Lange: "Molecular Cytology of Human Telomeres--Telomere Protein"
Scripps Research Institute Jean-Louis Reymond: "Catalytic Antibodies for Synthesis"
Stanford University Daniel T. Stack: "Dioxygen Activation by Model Copper Complexes"
State University of New York, Stony Brook Steven E. Rokita: "Probing Guanine Structure in Nucleic Acid Folding"
Texas A & M University Health Science Center Roderick E. Macallum: "Genetics and Biochemistry of Plasmid Transfer"
University of California, Los Angeles Robert P. Gunsalus: "Regulation of the E. coli Cytochrome Oxidase Genes"
University of California, Riverside Christopher Y. Switzer: "Synthesis and Evaluation of Novel Antisense DNA Analogs"
University of Chicago Augustine C. Kong: "Methods and Theory for Linkage Analysis"
University of Connecticut Health Center Juris B. Ozols: "Isolation and Structure of Microsomal Membrane Proteins"
University of Florida John P. Aris: "Nucleolar Function and Cell Growth in Yeast"
University of Illinois at Chicago Karl W. Volz: "Molecular Structure Studies of Bacterial Signal Proteins"
University of Iowa Rodney N. Nagoshi: "Regulation of Sex Specific RNA Splicing"
University of Michigan Daniel G. Remick: "Cytokines and Sepsis and Trauma"
University of Nebraska, Lincoln David S. Hage: "Chromatographic Automation of Immunoassays"
University of New Mexico, Albuquerque Mark R. Ondrias: "Electron Transfer Dynamics in Heme Proteins"
University of North Carolina, Chapel Hill Moo J. Cho: "Cytoplasmic Delivery of Oligonucleotides"
University of Pennsylvania John M. Murray: "3D Structure of Mitotic Microtubules"
University of Rochester George L. McLendon: "Electron Transfer Between Heme Proteins"
University of Southern California Miriam M. Susskind: "Interacting Regulators of Gene Expression"
University of Texas, Austin Sean M. Kerwin: "Shape Selective DNA Minor Groove Ligands"
University of Utah Elizabeth A. Leibold: "Iron Regulation of Gene Expression"
University of Virginia, Charlottesville Carol A. Otey: "Regulation of Focal Adhesion Structure"
University of Wisconsin, Madison Alan C. Rapraeger: "Heparan Sulfate and FGF Action"
Vanderbilt University Todd R. Graham: "Compartmental Organization of the Yeast Golgi Complex"
Washington University John A. Cooper: "Actin Cytoskeleton of Yeast"
Worcester Foundation for Experimental Biology Joel D. Richter: "Polyadenylation and Translational Control"
Yale University Lynne J. Regan: "Structure, Function & Folding of an RNA Binding Protein"
Yeshiva University Steven C. Almo: "Ribonuclease Superfamily--Structure-Function Studies"
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