On August 10th, Science published the results of Shi Yigong's team online, and for the first time analyzed the structure of the complex formed by human polycystin 1 and polycystin 2 with a resolution of 3.6 (A, equivalent to 10). -10 m).
Mutations in polycystin in humans can cause polycystic kidney disease, and about 1-2.5 people per 1,000 people are sick, which is more common. In 2003, a paper titled "Hot Problems in Autosomal Dominant Polycystic Kidney Disease Research" showed that the research focus of the disease lies in the structure of the disease-causing gene expression products, namely polycystin 1 and polycystin 2. With the function, subcellular localization, etc., this can clarify the molecular pathogenesis of polycystic kidney disease, and is expected to achieve a complete clinical cure.
After 15 years, the fine structure of the protein family was finally solved, and the "year-end question and answer" was completed. It is difficult to recognize that the protein family has a 4302 Amino Acid in polycystin 1 and contains 11 transmembrane helices, which is an ultra-complex protein. At the same time, polycystin 1 and polycystin 2 will complex and increase. The complexity of the structure.
System groping, get enough test usage
“The first step in resolving protein structure is to obtain a sufficient amount of protein with uniform properties.†On August 11, Dr. Wang Tingliang, one of the authors of the paper, told the Science and Technology Daily reporter that the preparations before “on the mirror†took five years.
For structural biologists, test proteins are often "stretched" in quantity. Protein is a human protein that is difficult to extract directly from human tissue. The research team cloned the gene in prokaryotes (such as E. coli) by known gene sequences, obtained a large number of gene copies, and then transferred to the commonly used tool cells for expression by virus infection to enrich the target protein.
In order to allow foreign genes to be expressed in the “new environmentâ€, the research team performed a lot of repetitive work to find the optimal expression system for the target genes (PKD1 and PKD2). "If the optimization of gene coding, the expression level of the protein can be increased, and the recombinant protein can be obtained in vitro," said Wang Tingliang.
At this point, the target protein is still in the cell, and the next step is to "fish" the target protein from the complex cells. A large number of extraction conditions, such as trying and screening a large number of detergents, determining protein boundaries, etc., can form a balance between quantity and quality of "a good kill one, no leakage net one thousand". In the end, the research team obtained precious and rare target proteins. "50 liters of cells can only extract about 100 micrograms of protein, only enough to complete 3 to 5 'on-the-lens' preparation, and the number of samples that meet the requirements of electron microscopy data collection is often only one or two." Wang Tingliang said.
Ants move, "reverse pushing" high-precision three-dimensional structure
The cryo-electron microscopy achieves a level A precision of the protein structure by emitting electrons and measuring the change in the trajectory of the electrons through the protein molecules. The field of view of each scan is very small. Therefore, after the protein is "on the mirror", it takes a long time to operate and a huge amount of information processing. For proteins with very large structures, the researchers will collect a large amount of data with an increase in multiples, and then perform structural reconstruction by calculation.
The three-dimensional structure of the whole protein is shaped like an ant moving. After obtaining a large number of multi-angle two-dimensional images, the image data is calculated and reconstructed by the later three-dimensional reconstruction system, and a schematic diagram of the three-dimensional structure of the protein is formed through the visualization system.
Through a cryo-SEM scan, Shi Yigong team obtained a unique one-to-three complex structure formed by polycystin 1 and polycystin 2, and further analyzed the structure and biochemical data.
"Face" is clear, and proposes a new perspective on the pathogenesis of polycystic kidney disease
The clear face of the protein complex gives the hypothesis an "iron certificate" or "reversal." The research team found a number of phenomena that contradicted the current mainstream academic views. Su Qiang, the first author of the paper, explained, “There is always controversy about whether these two proteins form calcium channels in the field of polycystic kidney research. The analytical structure does not support the hypothesis that the complex is a calcium channel.â€
The complex structure also refreshes the understanding of structural biologists on the similar folding "phase" of voltage-gated ion channels. Most of the ion channels previously reported are similar to folded structures or homotetramers, or single-stranded pseudo-quadruplexes, which is a one-to-three complex of two proteins.
"Interestingly, one of the proteins alone can form a tetramer. This unique structure means different working mechanisms and modes of regulation, enriching the understanding of structural biologists on the assembly of voltage-gated ion channels like folded proteins. "Shu Qiang said.
Fourteen years ago, in the "problem" paper, there are four "collaboration" models for polycystin 1 and polycystin 2. As the structure is clear, these hypotheses will soon have clear answers to the mechanism of polycystic kidney disease. Research brings a useful reference. (Reporter Zhang Jiaxing)
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