Study On The Mechanism Of Conformation Changes Of Cyclic Cyclic Adenosine Receptor

Conformational changes play pivotal role in the protein functions. In this work, various techniques have been used to investigate the allosteric mechanism of cAMP receptor protein (CRP) and pyruvate kinase (PK).cAMP receptor protein (CRP), also referred as Catabolite gene activator protein (CAP), is a classic transcription regulator in prokaryotes. CRP is a dimer made up of identical subunits, each one consists of two domains:a larger N-terminal3’-5’cAMP binding domain and a smaller C-terminal DNA binding domain. After binding the cAMP, CRP undergoes an allostery and recruits the DNA promoters, then actives the RNA polymerase to initiate transcription of many catabolite genes. CRP has become an important paradigm for allostery. The crystal structures of CRP-cAMP, CRP-cAMP-DNA, and CRP-cAMP-DNA-RNAP complex have all been determined. On the basis of structural information, the DNA recognition helix (F-helix) is buried between the two DNA binding domains in the absence of cAMP and cannot interact with the DNA. cAMP binding triggers large conformational changes leading to F-helix exposed to the surface, which enable the protein to recognize specific DNA sequences. Although people have certain understanding to handle the knowledge of the allosteric mechanism of CRP, there are many issues to be solved. For xample, many structural information of apo-CRP, CRP-cAMP2, CRP-cAMP3and CRP-cAMP4have been obtaind, but the structure of CAP-cAMP1complex structure remains unknown. The conformation of apo-CRP is very different from that of CRP-cAMP2, which makes the studies of CAP-cAMP1very essentially.In addition, Pyruvate kinase (PK) is an important glycolytic regulatory enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate in the presence of monovalent and divalent cations. RMPK is a homo-tetramer consisting of four chemically identical subunits, each folding into four domains:N-terminal domain, domain A, domain B, and domain C. The four subunits of PK form two distinct subunit interfaces, namely the Y-interface and the Z-interface. Three tryptophan residues are present per PK monomer:Trp-157is in domain B and close to the active site, whereas Trp-481and Trp-514are in domain C and close to the Y-interface. Therefore, those intrinsic tryptophan residues are sensitive probes for monitoring protein conformational changes induced by the binding of various ligands. The domain B is highly mobile. The inactive state of PK can be represented by a rotation of the domain B in opening of the cleft between the B and A domains, and the active state can be represented by closing of the cleft. The regulatory behavior of PK can satisfactorily be described by the two-state model of Monod-Wyman-Changeux. However, the catalytic allosteric regulation of PK has has remained elusive.In this thesis, totally five chapters of this dissertation are discussed as follows:In the first chapter, a brief introduction on the research and development of cAMP receptor protein (CRP) and pyruvate kinase (PK) was described. Then a scheme of research basing on the background information was proposed. Various techniques, such as crystallography, spectroscopy and biophysics were used to study the conformational changes mechanism of CRP and PK.In the second chapter, the expression, purification, crystallization and the crystal structure of the two different cAMP binding domains of CRP were introduced. In this work, two specific proteolytic enzymes, subtilisin and chymotrypsin, had been used to digest WT-CRP. Two different cAMP binding domains of CRP, namely S-CRP (residues1-133) and CH-CRP (residues1-137) were obtained. A special method had been used to obtain the crystal of S-CRP-cAMP and CH-CRP-cAMP, and their structure were determined at a resolution of2.0A and2.8A, respectively. This research focused on the crystal structure of S-CRP-cAMP. Each asymmetric unit of S-CRP-cAMP contains four dimmer. The overall fold of S-CRP-cAMP and the basic dimerization way are similar with those in the full-length CAP. Interestingly, according to the number and situation of cAMP binding, the four dimers can be classified into three types, namely, S-CRP-cAMP2、S-CRP-cAMP1and S-CRP-cAMP2,.Moreover, S-CRP-cAMP1and S-CRP-cAMP2> had been obtained for the first time. It is proposed that one cAMP binding is enough to switch CAP activity in vivo and the second cAMP binding situation doesn’t influent the rearrangement of the dimer C-helices (residues112-133).In the third chapter, the conformational and structural dynamics changes of the two different cAMP binding domains of CRP in the presence and absence of cAMP were introduced. In order to confirm the role of the hinge (residues134-138) and the interaction of loop3(residues53-57) and Phe136in the conformational changes and the signal transmission, Fourier transform infrared (FT-IR) spectroscopy had been used to study the secondary structure contents and the dynamics changes of S-CRP and CH-CRP in the presence and absence of cAMP. The results reveal that CRP activation upon cAMP binding involves significant changes in secondary structures, an increase in C-helix and a decrease in either the β strand4or p strand5, and both S-CRP-cAMP and CH-CRP-cAMP shift to less dynamic conformations, evidenced by slower amide H-D exchange rates. The hinge range increases the dynamic behavior of CH-CRP and CH-CRP-cAMP, whereas the interaction of loop3and Phe136in the hinge region of the adjacent subunit does not involve in the cAMP-induced allosteric signal transmission.In the fourth chapter, the conformational changes mechanism of PK induced by the various ligands were introduced.In this study, three tryptophan residues had been site-specific mutated, respectively. Fluorescence quenching, Circular dichroism (CD) spectroscopy and FT-IR spectroscopy were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Mutation of the tryptophan residues did not elicit any observable difference in the secondary structure of PK. Meanwhile Trp157accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481and Trp-514are brought into an aqueous environment after phenylalanine binding. We focused on the role of domain B in the catalytic allosteric reaction of PK. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft.In the fifth chapter, the thermodynamic characterization of the allosteric regulation of PK was introduced. The thermodynamic parameters of the catalytic reaction of PK were firstly obtained by isothermal titration calorimetry (ITC), which played an important role in confirming the effects of the different activating cations (K+and Mg2+) and inhibitor (Phenylalanine) on the conformational changes of PK. The percentage of the active state increased with increasing concentration of K+or Mg2+, whereas increasing Phe concentration had the opposite effect. Especially, it is hypothesized that the activation of RMPK involves two processes. First, the interaction of Mg2+leads to a more exposed active site of RMPK. The process is rapid and only a small quantity of Mg2+can make RMPK transform to the intermediate state. Second, the subsequent binding of K+causes a critical orientation of the active site, which plays a more decisive role for PEP binding to PK than Mg2+.