Scientists are harnessing hard X-rays in the fight against cancer. A team of researchers, in conjunction with the U.S. Department of Energy’s (DOE) Argonne National Laboratory, has used ultrabright X-ray light to determine how specific types of antibodies can tell the difference between different forms of a cancer-linked molecule. These new insights will help scientists design better antibodies for potential treatments.
Tony Hunter, professor at the Salk Institute for Biological Studies, led this new research, building on years of study at his lab into amino acids, the building blocks of proteins. Hunter and his team were the first to show that adding phosphate to tyrosine, one of 20 amino acids in the human body, contributes to the progression of cancer. Their discovery not only led to the development of anticancer drugs, but also inspired researchers to start examining phosphate in combination with other amino acids.
“Our antibodies are going to be key to studying this relatively understudied process of histidine phosphorylation, and thanks to X-ray crystallography, we now know how they work. This means we can potentially improve them for specific purposes and even perhaps for use in the clinical arena where we see evidence that histidine phosphorylation plays a role in disease.” — Tony Hunter, professor, Salk Institute for Biological Studies
Histidine, an amino acid the body uses to synthesize proteins, is the new target under study at the Hunter Lab. When phosphate is added, it forms phosphohistidine, an unstable molecule that has been linked to liver and breast cancer and neuroblastoma, a type of cancer often found in the adrenal glands.
To better understand phosphohistidine’s potential role in cancer, the Hunter research team has, for the past eight years, been developing and studying antibodies that can bind to it. But to discern exactly how these antibodies work, they needed a more specialized set of tools.
The Advanced Photon Source (APS), a DOE Office of Science User Facility at Argonne, was one of three light source facilities the research team used to gain more insight into this problem. At the facilities, they used a technique known as X-ray crystallography to determine the crystal structures of their antibodies bound to peptides (short amino acid sequences) containing phosphohistidine. Their work and findings were recently published in the Proceedings of the National Academy of Sciences (PNAS).
“Our antibodies are going to be key to studying this relatively understudied process of histidine phosphorylation, and thanks to X-ray crystallography, we now know how they work,” said Hunter “This means we can potentially improve them for specific purposes and even perhaps for use in the clinical arena where we see evidence that histidine phosphorylation is connected to cancer.”
To make use of this technique, Hunter’s team worked alongside researchers at The Scripps Research Institute and used three different light sources — the APS, the Advanced Light Source at DOE’s Lawrence Berkeley National Laboratory, and the Stanford Synchrotron Radiation Lightsource at DOE’s SLAC National Accelerator Laboratory. All three are DOE Office of Science User Facilities that provide extremely intense, small X-ray beams that are particularly useful for this type of technique.
Researchers first grew crystals of their antibodies bound to phosphohistidine peptides. These were then sent to the light sources, which had capabilities that allowed the researchers to place their crystals in an X-ray beam remotely. Upon contact with the crystals, the beams scattered, creating diffraction patterns that were collected and used to determine the 3-D atomic structure of the antibodies combined with the phosphohistidine peptides.
“X-rays have wavelengths that are about the size of atoms, and they scatter strongly. But it’s not so easy to make a lens that can recombine these rays to form an image near atomic resolution,” said protein crystallographer Michael Becker of Argonne’s X-ray Science Division. “So instead, researchers collect diffraction data on detectors and use mathematics, physics and chemistry in the computer to essentially calculate an image of the molecule in the crystal.”
X-ray crystallography allows scientists to determine the molecular and atomic structure of these tiny crystals. By measuring these diffracted beams, scientists can reconstruct an image of the atoms and their position in the sample, as well as a host of other information.
“What crystallography did was enable us to look at atomic interactions between the antibody and the antigen, which in this case was the phosphohistidine,” said Ian Wilson, a structural biology professor at The Scripps Research Institute and a co-author on the paper.
The resulting insights not only advance our understanding of phosphohistidine’s potential role in cancer, but can also help other scientists looking to design better antibodies to suit their own research purposes.
“From the data, we learned how small differences in atomic interactions help the antibodies to differentiate the two different isoforms of phosphohistidine, and also how these antibodies are able to recognize different peptides which undergo histidine phosphorylation,” said Rajasree Kalagiri, a Salk postdoctoral researcher and lead author of the study.
The study, titled “Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies,” was published in PNAS on February 9.
Additional authors of the study include Jill Meisenhelder and Stephen R. Fuhs of the Salk Institute; Robyn Stanfield of The Scripps Research Institute; and James J. La Clair of the University of California San Diego.
The Advanced Photon Source is operated for the DOE Office of Science by Argonne National Laboratory. X-ray crystallography at the APS was performed at the General Medical Sciences and Cancer Institutes Structural Biology Facility which has been funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396).
Use of the Stanford Synchrotron Radiation Lightsource was supported by DOE Office of Science’s Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The Advanced Light Source is a DOE Office of Science User Facility under Contract DE-AC02-05CH11231. This project was also funded by the Leona M. and Harry B. Helmsley Charitable Trust and the Skaggs Institute for Chemical Biology at The Scripps Research Institute.
About the Advanced Photon Source
The U. S. Department of Energy Office of Science’s Advanced Photon Source (APS) at Argonne National Laboratory is one of the world’s most productive X-ray light source facilities. The APS provides high-brightness X-ray beams to a diverse community of researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. These X-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being. Each year, more than 5,000 researchers use the APS to produce over 2,000 publications detailing impactful discoveries, and solve more vital biological protein structures than users of any other X-ray light source research facility. APS scientists and engineers innovate technology that is at the heart of advancing accelerator and light-source operations. This includes the insertion devices that produce extreme-brightness X-rays prized by researchers, lenses that focus the X-rays down to a few nanometers, instrumentation that maximizes the way the X-rays interact with samples being studied, and software that gathers and manages the massive quantity of data resulting from discovery research at the APS.
This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
Argonne National Laboratory seeks solutions to pressing national problems in science and technology. The nation’s first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state and municipal agencies to help them solve their specific problems, advance America’s scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy’s Office of Science.
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