Study On The Structure And Function Of Cyclic Adenosine Receptor Protein

The Escherichia coli cyclic AMP receptor protein (CRP), also referred to as catabolite gene activator protein (CAP), plays an important role in the transcriptional regulation. Upon cAMP binding, CRP adopts a conformation that promotes its interaction with specific DNA sites. After the interaction, CRP recruits RNA polymerase (RNAP) to the promoter to ultimately regulate the transcription of more than 200 genes. Of the transcription regulatory proteins, CRP was the first to purified, and the first to have its 3D structure discovered. So the mechanism of cAMP-dependent CRP activation is perceived as a classical model for allosteric transition.The conformation of CRP can be categorized into three groups. The first group, designated as "off" state, represents the ligand-free, inactive conformations; The second group depicts a fully active, namely "on" state, represents the ligand-free, active conformations; The other structures are designated as "intermediate" state, represents the ligand-bound, inactive and the ligand-free, active conformations. All of the twenty-four CRP structures in PDB are regarded as "on" state except for apo-CRP and D138L CRP, which belong to "off" state. However, the 3.6 A apo-CRP is too low to describe the details in the structures. None of the structures is reported as CRP intermediate state. Therefore, it is difficult to accurate understand the allosteric mechanism and process for CRP. In addition, the fifteen structures containing DNA, all of the dsDNA are cracked, have a 4-bp self-complementary overhang on the 3’end, which hybridize with each other to provide completely symmetric binding sites to each molecule of the protein dimer. Thus, the question needs to be answered:can two symmetrical half-site sequences really replace full-length fragments?In this study, some CRP structures had been determined by the means of experiences in molecular biology and structural biology. The analysis for the structures is beneficial for understanding the details of the CRP structures and its allosteric mechanism.Wild-type CRP was expressed and purified. The crystals structure of the apo-CRP had been determined at a 2.5 A resolution. The space group belonged to P21 and four CRP molecules in the asymmetric unit. Compared with the previous apo-CRP structure (PDB ID:3HIF), with a resolution of 3.6 A, showed two main differences. First, there exited a short a-helix in the β4-β5 hairpin region, it was consistent with NMR datas. Meanwhile, the hinge region exhibited difference on the lengths and the conformations. The hinge region of CRP should be composed of amino acids T127 to D138 rather than V131-D138 defined in 3HIF nor L134-D138 defined in previous. It was consistent with the PrfA, another member of CRP-family proteins. The structure indicates that even though in the absence of cAMP, external stimulus occurs or intracellular physiological environment alters, may cause the conformation of apo-CRP transforming overall. The adjustments not only include the CTD direction relative to the NTD, but also the conversion between random coil and a-helix.A complex crystal structure which contains one CRP molecule, one cAMP and a half 38-bp full-length dsDNA fragment in the asymmetric unit was obtained at a 2.9 A resolution. The crystals belonged to the space group P3121. Although DNA density was not complete, it was clear for the 22-bp consensus sequences in the DNA. This structure was similar to the CRP-cAMP-DNA (half-site) (PDB ID: 1ZRC). The results indicate that when the sequence is completely identical, the half-site DNA used in 1ZRC can replace the full-length DNA. In addition, it confirms that the fifteen structures contain half-site DNA can represent the real conformations.A series of CRP mutants, D53H, S62F, G141S, G141R, G141K, G141D, R142C and L148R were constructed. These mutants which known as CRP* can activate CRP-dependent promoters in the absence of cAMP. They represent the intermediate state. The crystals of D53H, S62F and G141D were obtained, and the structure of D53H CRP was determined at a 2.9 A resolution. It belonged to the space group P212121. The asymmetric unit was found to contain eight CRP molecules. As the first reported intermediate state, the structure was different from apo-CRP and cAMP-CRP. As the DNA-binding domains did not overturn, its conformation can be considered as the initial conformation during the allosteric process. It was completely opposite to the viewpoint that the CRP* structures were similar or even equal to the cAMP-CRP in previous.The complex crystals of the ligand-free D53H CRP-DNA (half-site) were obtained. The structure was determined at a 3.4 A resolution, which belonged to the space group P212121. There were two CRP molecules in each asymmetric unit. The election density was continuous for the main chains and DNA. With DNA binding, the structure showed similar to cAMP-CRP conformation, which represented as "on" state, even though the D53H CRP conformation is different from the cAMP-CRP. The structure not only supports the view that D53H CRP conformation is an intermediate state, but also indicates that there may exist synergistic effects between CRP and DNA in the allosteric process.According to the four structures in the study and other structures in PDB, a new model of allosteric mechanism was established, and more details of allosteric process were expounded. Upon cAMP binding, the dynamic balance of apo-CRP with the "off" state was broken, local variations amplified through the signal network step by step, resulted in allosteric effects overall. On the initial stage of allosteric, CRP conformation showed loose, the two DNA binding domains moved to the same side in the form of one rigid body. The hinge region was disordered and extended upward to provide enough energy and space for the allosteric. E a-helix and F a-helix (the fifth and sixth a-helix of CRP) recognized the specific DNA, and synergistic allosteric with DNA. With the deepening of the process, the C-terminal domains overturned overall, then DNA bound, CRP conformation tends to a new equilibrium, C a-helix extended and two subunits interacted to stable "on" state. This allosteric model is general close to the view that local perturbation driving the global transition.