Just one gene – and a whole bunch of diseases
The XPD protein is a component of the mechanism needed to repair DNA damage. The uniqueness of XPD lies in the fact that mutations in different parts of its gene underlie three human diseases: pigmented xeroderma, characterized by hypersensitivity of the skin to light and leading to the development of cancer; Coccain syndrome, combining growth disorder, retinitis pigmentosa, optic nerve atrophy, deafness, mental retardation and premature aging; and trichotiodystrophy (congenital ichthyosis) is a form of premature aging characterized by brittle hair and scaly skin.
Scientists of the Lawrence Berkeley National Laboratory, part of the US Department of Energy, and the Scripps Research Institute, working under the leadership of Professor John Tainer, were the first to decipher the structure of the XPD protein. The data obtained explain how violations of the structure of this protein, even such minor ones as changes in two adjacent amino acid residues, can lead to the development of diseases with various symptoms, and provides new information about the processes of aging and cancer development.
The XPD protein is part of the TFIIH molecular complex responsible for the recovery of nucleotides that have fallen out of the DNA sequence for one reason or another. TFIIH is also involved in normal gene transcription and repair of associated damage. Such repair requires the elimination and replacement of a fragment of a DNA chain containing one or more damaged nucleotide bases, recognized by violations in the helical structure.
TFIIH connects to the site of damage, and the helicase enzymes included in its structure, one of which is XPD, unwind a fragment of 30 nucleotides long containing the damage. After that, a whole complex of enzymes enters the process: the XPG and XPF nucleases cut out the damage, the polymerase restores the correct sequence in accordance with the complementary chain, and finally the ligase connects the ends of the restored chain.
Despite the fact that the XPD protein is not present in all organisms (bacteria use a different mechanism to repair similar damage), the structure of the gene encoding it, as well as other genes responsible for the basic functions of living organisms, is similar in many species, from humans to prokaryotes of the Archaea family, often living in extreme conditions. conditions, for example, in hot springs. The sequence of this gene has been known for a long time, but the structure of the protein could not be deciphered for a long time due to the complexity of its purification and crystallization necessary for X-ray crystallography.
At first, the authors tried to work with the human version of XPD, but it turned out that it is very poorly soluble in water and unsuitable for crystallization. In search of a homologous protein, scientists have traditionally turned to thermophilic organisms living in hot springs, whose proteins are usually the most stable.
Belonging to the archaea family, Sulfolobus acidocaldarius lives at a temperature of 80 degrees Celsius and pH = 3, which corresponds to life in a container with hot acid. The variant of the XPD protein belonging to this organism is really stable and, despite the absence of protruding structures characteristic of a human protein in its structure, its catalytic core is very similar to the core of a human enzyme. In addition, 22 of the 26 fragments of the human XPD gene sequence in which disease-associated mutations occur were found in the corresponding S.acidocaldarius gene.
A unique feature of the structure of XPD and a number of other helicases is a group of iron and sulfur atoms that easily interacts with oxygen. To prevent oxidation of this group, the purified protein must be crystallized in an anaerobic chamber. The brown-red color of the grown protein crystals indicates the unchanged state of the iron-sulfur group.
Two protein forms were used for X-ray crystallography: a native conformation and a variant in which all the amino acid residues of methionine were replaced by selenomethionine residues. Heavy selenium atoms are clearly visible in the synchrotron beam and make it possible to determine the position of methionine residues in the amino acid sequence with high accuracy.
Of the four domains of the protein molecule, two, HD1 and HD2, are typically helicase sequences, one is the 4FeS iron-sulfur domain, the protruding shape of which indicates its participation in the registration of damage and the opening of the DNA helix. The fourth domain – Arch – turned out to be a complete surprise. Together with 4FeS, Arch forms an unusual arch in the XPD globule over a tunnel-like hole at the end of a long chute. The electric charge of the amino acids lining the gutter and the relief of the structure indicate that it is a corridor for binding and moving DNA.
The figure shows four XPD domains: HD1 (green) and HD2 (blue) helicases, 4FeS iron-sulfur complex (brown-red) and Arch (purple). The sites of mutations that cause pigmented xeroderma are marked in red; pigmented xeroderma in combination with Coccain syndrome is golden, and trichotiodystrophy is purple.
After finding out the structure of the protein molecule, the authors tested its following functions in laboratory conditions: the ability to hydrolyze ATP – ATPase activity, the ability to unwind the DNA chain – helicase activity and the ability to bind DNA.
The researchers evaluated the functions of 15 mutant forms of XPD containing specific mutations associated with xeroderma pigmentosa, Coccain syndrome and trichotiodystrophy. All three diseases increase the sensitivity of the skin to ultraviolet sunlight – the main cause of nucleotide damage eliminated by the TFIIH complex. However, pigmented xeroderma increases the risk of developing skin cancer thousands of times, and the other two diseases do not affect this indicator, but lead to various forms of premature aging.
The effects of individual mutations are very difficult to predict without knowledge of the molecular structure of the protein. In the case of XPD, it remained a mystery to scientists how very similar mutations (in one case, a change in one or the other of two adjacent amino acids) could cause different diseases.
According to the authors, only having an idea of the structure of the protein molecule, it is possible to understand how mutations of neighboring amino acids can have various effects: one changes the properties of the DNA-binding channel, and the other changes the movement of domains relative to each other. In one case, a person develops pigmented xeroderma, and in the other – Coccain syndrome.
The mutations causing the pigment xeroderma directly affect the DNA and ATP binding channels inside the XPD molecule. These changes reduce the efficiency of DNA unwinding by mutant forms of protein.
Mutations of another type, which cause Coccain syndrome in combination with pigmented xeroderma, disrupt the mobility of helicase domains, which should change the conformation of the molecule in the process of repairing DNA damage.
Mutations lead to trichotiodystrophy, which may or may not reduce the activity of helicase, but invariably disrupt the stability of the entire structure. Some mutations of the 4FeS domain disrupt the protein structure so much that they completely suppress helicase activity.
Deciphering the structure of the XPD catalytic core is a significant achievement in understanding the mechanisms of transcription and the repair of DNA damage provided by TFIIH and provides information on how various violations of these mechanisms cause signs of physical aging and lead to the formation of cancer. However, scientists need to learn a lot more about this extremely important protein.
Evgeniya Ryabtseva
Portal "Eternal youth" www.vechnayamolodost.ru based on the materials of ScienceDaily
05.06.2008