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Paper of the Month: The Structure of Lombricine Kinase Share This OHSU Content

05/17/11  Portland, Ore.

JBC-cover-march2011This month's featured paper is from the Journal of Biological Chemistry, and is titled “The Structure of Lombricine Kinase: Implications for phosphagen kinase conformational changes.”

This research was conducted as part of an investigative collaboration, and included the Department of Biochemistry and Molecular Biology’s Olga Kirillova, PhD, Omar Davulcu, PhD, Qing Xie, PhD, Michael Chapman, PhD, and colleagues.*

Creatine kinase is the enzyme charged with maintaining steady concentrations of our cellular energy currency, adenosine triphosphate or ATP. It does this by catalyzing the transfer of a phosphate group to and from creatine, a dietary supplement that athletes hope will enhance their ability to respond to bursts of activity. Medically, creatine kinase levels are measured in the clinical diagnosis of recent heart attack and stroke, because its physiological role requires large amounts of the enzyme in the heart and brain, and its release from damaged cells can be detected in serum.

Instrumentation to support this research and related projects was recently provided by the first award from the OHSU Emerging Technology Fund.

Structural biology has become a cornerstone of biomedical science, the atomic structures of biomolecules providing a framework for researchers to understand function and mechanism at a molecular level, and a foundation in the basic science education of medical professionals. Metabolic enzymes were among the first targets for structural biology, but by the mid 1990s, this enzyme had not yielded to seven independent assaults on its structure.

paper_researchers_photo_51611Genomics opened new avenues, and the Chapman group hoped that an indirect approach might be more fruitful. Invertebrates have a simpler form of the enzyme that catalyzes an analogous reaction and is thought to be the evolutionary ancestor for the enzyme family. It was cloned from horseshoe crab, expressed and its structure was solved by x-ray crystallography. By this time a structure for chicken creatine kinase had been solved in Germany, but unique insights were available to the Chapman group, because they had crystallized the enzyme in its reactive state as a transition state analog, and determined the structure at exceptionally high resolution, leading to complete revision of the enzyme mechanism.

Biochemists want to understand how enzymes increase the rate of reactions, and there is a new appreciation that proteins are not static structures. Jeremy Knowles, Biochemist and late Dean at Harvard, remarked that “studying the photograph of a racehorse cannot tell you how fast it can run”, and thus there is a limit to what a snapshot protein structure can reveal. Creatine kinase and its relatives undergo large changes in structure to wrap around the substrates, eliminating water and the possibility of a wasteful side reaction. Early work suggested that the binding of ATP induced most of the changes, and a few recent exceptions were interpreted as evidence of allosteric (or remote) regulatory control.

“With lombricine kinase, evolutionary diversity is put to use again,” said Michael Chapman, PhD, Professor, Department of Biochemistry & Molecular Biology. “This enzyme from an annelid worm was of interest because of its decreased substrate specificity, but when the structure of its nucleotide-bound complex was determined, it was intermediate between substrate-free and –bound structures of other members of the family.” By comparing structures from different organisms, the scientists in the Chapman lab realized that there was a continuum of structures rather than a single nucleotide-bound form.

Returning to arginine kinase, the group reported, in the January issue of the Journal of Molecular Biology, a combination of x-ray crystallography and NMR Residual Dipolar Coupling measurements demonstrating that even without substrates, in solution, the enzyme exists as a dynamic equilibrium of the open and closed (substrate-bound) forms seen crystallographically. Furthermore, in the May issue of Biochemistry, they report measurements of the enzyme kinetics and NMR Relaxation Dispersion over a range of temperatures that establishes that the protein conformational changes are the rate-limiting step in the enzyme-catalyzed reaction. “The Lombricine Kinase structures complete the picture by showing that ATP-binding does not force the enzyme into pre-determined structure, but shifts an equilibrium to a finely balanced and highly dynamic state,” said Dr. Chapman.

Why so much attention on these enzymes? Dr. Chapman points out that “no other representative enzyme” has proven amenable to the combination of high resolution crystallography and NMR spectroscopy needed to extend our understanding beyond static structure to the functionally important dynamics. “Thus, this system offers a window on how one might predict many other proteins to behave,” he said.

lk_imageFIGURE 1: Enzymes like creatine kinase and arginine kinase close around the substrates upon binding. The ribbons show the protein structure in open (red) and closed (blue) configurations. The substrates can be seen as multi-colored stick molecules in the center.

 

 

 

 

 

 

 

Pictured (l to r): Qing Xie, PhD; Omar Davulcu, PhD; Michael Chapman, PhD; Olga Kirillova, PhD

* From the Department of Chemistry & Biochemistry, Florida State University, D. Jeffrey Bush, PhD, and Shawn Clark, PhD; From the Department of Biological Science, Florida State University, W. Ross Ellington, PhD; From the Institute of Molecular Biophysics, Felcy Fabiola, PhD, and Thayumanasamy Somasundaram, PhD; From the Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Olga Kirillova, PhD, Omar Davulcu, PhD, Qing Xie, PhD, and Michael Chapman, PhD.

 
Emerging Technology Fund at work

The OHSU Emerging Technology Fund (ETF) makes funds available to faculty who need to purchase equipment or technology for state-of-the-art research. Instrumentation like that recently funded by ETF played an important role in the findings of this paper.

Dr. Chapman, recipient of the first ETF award, said “Instrumentation is being ordered that will replace equipment in service for 14 years, well beyond its lifetime. It will support the determination of protein structures at OHSU, and also be compatible with the modern practice of screening samples before shipment to one of several National Laboratories for particularly high resolution characterization.” OHSU’s Department of Biochemistry & Molecular Biology is a member of the Molecular Biology Consortium which runs an experimental station at the Advanced Light Source at the Lawrence Berkeley National Laboratory. The new instrumentation will be both a stand-alone resource and will support local sample optimization to maximize the return on access to shared high intensity x-ray sources like that in Berkeley. 

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ABOUT THE PAPER OF THE MONTH

The School of Medicine newsletter spotlights a recently published faculty research paper in each issue. The goals are to highlight the great research happening at OHSU and to share this information across departments, institutes and disciplines. The monthly paper summary is selected by Associate Dean for Basic Science Mary Stenzel-Poore, PhD. Here’s what Dr. Stenzel-Poore had to say about this paper:

“This paper is a beautiful example of how studies in structural biology contribute to our understanding of the specific functions of important cellular molecules and enhance our view of comparative molecular evolution among related molecules. The experimental observations made here require sophisticated instrumentation for x-ray diffraction studies. The Chapman group, along with a group of OHSU scientists with research interests in structural biology, were the first recipients of an OHSU Emerging Technology Fund Award to acquire new state-of-art instrumentation to determine protein structure and perform x-ray diffraction analyses.”