The Preliminary Crystallization Study Of The Protein STING

STING is an important adaptor protein in regulating the production of type Ⅰ interferon of the innate immune system. It functions in the innate immune responses caused by bacterial, virus and eukaryotic pathogens[1. Recently researches found out that STING functions as a direct immune sensor of bacterial second messenger c-di-GMP[13]. STING was previously found to be a trans-membrane protein containing five N-terminal trans-membrane a-helix (1-173aa) and a C-terminal soluble domain (174-379aa).The N-terminal domain of STING may be associated with its sub-cellular distribution, while the C-terminal domain functions mainly by interacting with other molecules. There is no homologous protein structure of STING in the PDB databank, which suggests that it may have a unique protein structure. To elucidate the mechanism of how STING senses the c-di-GMP signal, we conducted the the preliminary crystallization studies of the protein STING.At first, we got the soluble C-terminal domain of STING (149-379aa, called STINGCTD) by the expression of its different segments. Then we did the crystal screen using the sitting-drop methods and got the crystal of STINGCTD in buffer containing. After that we optimized the crystal through the following different aspects:(1) design different gradients of PH and precipitant of the buffer;(2) conduct the additive and detergent screen;(3) decrease the temperature of the crystal growth;(4) dehydration of the STINGCTD crystal by lithium acetate. And eventually we got the single crystal that had a high x-ray diffraction resolution. The primary data processing of STNGCTD diffraction data shows that the crystal belongs to the space group C222i. One asymmetry unit contains one molecule of STINGCTD, which forms a dimer with its symmetry-related molecule. As there is no homologous protein structure of STINGCTD in PDB, we used its Se-Met labeled derivative to solve the phase problem in structure determination. At last, we got high diffraction resolution crystal of its Se-Met labeled derivative by seeding with the native ST1NGCID crystal. The crystal of SeMet-STINGCTD belongs to space group P21,each asymmetry unit also contains one molecule of STINGCTD, which forms a dimer with its symmetry-related molecule. Then we solve the structure of apo-STINGCTD by single wavelength anomalous diffraction. STINGCTD forms a dimer, which has a V-shaped architecture. The predicted fifth trans-membrane a-helix is actually part of the c-terminal soluble domain which participates in the formation of dimer interface The protein structure represents a new folding form.To elucidate the mechanism of how STINGCTD sensed (he c-di-GMP, we optimized the crystal of c-di-GMP bound STINGCTD complex Eventually we collected a set of dilTraction data of2.40A. The crystal of c-di-GMP bound ST1NGCTD complex belongs to space group P212121. Each asymmetry unit contains one molecule of c-di-GMP bound STINGCTD comple...