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Enzyme Structure Provides Insights into Cancer and Aging Print

XPD helicase is an enzyme that unwinds the DNA double helix; it is one component of an essential repair mechanism that maintains the integrity of DNA. XPD is unique, however, in that pinpoint mutations of this single protein are responsible for three different human diseases: in xeroderma pigmentosum (XP), extreme sensitivity to sunlight promotes cancer; Cockayne syndrome (CS) involves stunted growth and premature aging; trichothiodystrophy (TTD), characterized by brittle hair and scaly skin, is another form of greatly accelerated aging. At the ALS, researchers from Berkeley Lab and The Scripps Research Institute recently solved the structure of XPD. The structure gives novel insight into the processes of aging and cancer by revealing how discrete flaws—as seemingly insignificant as a change in either of two adjacent amino acid residues—can lead to diseases with completely different physical manifestations.

DNA: The Thread of Life

XPD is shown dividing DNA that is held by the three fates of Greek mythology: Clotho (left) spun the thread of life, Lachesis (bottom) measured it, and Atropos (right) cut the thread, determining life span. XPD opens damaged DNA to allow repair enzymes to excise the damaged patch. Figure design by Doug Ng and Michael Pique.

Using data collected at the SIBYLS Beamline 12.3.1, the researchers solved the crystal structure of XPD from Sulfolobus acidocaldarius. The data show that the catalytic core of XPD has four domains: two helicase domains named HD1 and HD2, an Arch domain, and an iron–sulfur cluster domain (4FeS). The Arch and 4FeS domains are insertions into the linear sequence of the HD1 domain, and these domains all lie in the same plane and form a flat, pentagonal-shaped box with a small hole. In the researchers' proposed model, DNA is unwound as it travels through the hole formed between the HD1, Arch, and 4FeS domains. HD2 protrudes from the box to form long channels between itself and the other three domains. These channels, according to the model, bind single-stranded DNA and ATP.

The solved crystal structure of XPD is shown with domains colored as follows: helicase domains HD1 and HD2 (blue and green), 4FeS (orange), and Arch (purple). DNA (pink) was computationally modeled into the structure. Spheres mark the location of residues associated with human disease-causing mutations: XP (red), XP/CS (gold), and TTD (purple).

Twenty-six single amino-acid changes in XPD can cause disease in humans. The position of each mutation along the linear sequence of XPD does not predict the large differences between XP, XP/CS, and TTD. In fact, even mutations in adjacent amino acids cause different diseases. By combining structural analyses of the wild-type protein with biochemical measurements of XPD mutant enzyme activities, the researchers now have a detailed molecular framework that begins to explain why a mutation at one amino acid leads to XP rather than XP/CS or TTD. Since XP mutations cluster along the DNA- and ATP-binding channels, they decrease helicase activity. Besides losing helicase activity as expected for XP mutations, XP/CS mutations also replace small flexible amino acids with larger ones, reducing flexibility. These mutations are largely found at the HD1–HD2 interface and therefore affect the conformational crosstalk between these domains to impact DNA-binding activity or potentially interactions with partner proteins. TTD mutations are found throughout the protein and disrupt stabilizing interactions between amino-acid side chains and/or protein main chains. These changes disrupt the framework of the XPD protein to reduce its structural integrity.

How do these changes in the XPD protein structure determine a patient's fate? Based upon XPD structural biochemistry, the researchers predict that an XP patient has reduced DNA repair capacity and therefore cannot repair all of the damage that occurs on sunlight-exposed areas of the skin. XP/CS patients have the same cancer susceptibility as XP patients with the added complication of a mutation that reduces XPD flexibility. This compromises the cell's ability to survive, leading to premature aging. TTD seems to work in an equal, but essentially opposite way. Instead of decreasing flexibility, TTD mutations increase protein flexibility, which likely disrupt the interactions of XPD with other proteins. These combined structural and biochemical results broaden our understanding of how XPD structural changes might have an impact on cancer or premature aging. Significantly, these results reveal the importance of the overall and detailed three-dimensional structure as well as the active site and interfaces in biological function.

Research conducted by L. Fan and A.S. Arvai, (The Scripps Research Institute); J.O. Fuss, Q.J. Cheng, M. Hammel, and P.K. Cooper (Berkeley Lab); V.A. Roberts (University of California, San Diego), and J.A. Tainer (The Scripps Research Institute and Berkeley Lab).

Research funding: National Institutes of Heath. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: L. Fan, J.O. Fuss, Q.J. Cheng, A.S. Arvai, M. Hammel, V.A. Roberts, P.K. Cooper, and J.A. Tainer, "XPD helicase structures and activities: Insights into the cancer and aging phenotypes from XPD mutations," Cell 133, 789 (2008).

ALSNews Vol. 295, February 25, 2009

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