Structure And Allostery Mechanism Of CAMP Ligated D138L Mutant Of CAMP Receptor Protein

The (?)AMP receptor protein (CRP) from Escherichia Coli is a classic transcription regulator in prokaryotes.CRP is a dimer made up of identical subunits, each subunit consists of two domains:the larger N-terminal domain (residues 1-137), which is predominantly (3-stranded, contains the cAMP binding pocket;the smaller C-terminal domain (residues 138-209), which is mainly populated by a-helix, contains the DNA binding motif. 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.Based on the studies of transcriptomics and bioinformatics, the transcriptions of nearly 200 different promoters are regulated by CRP. As one of the most studied transcriptional regulators, and the similar cAMP-and DNA-binding motif found in other important function proteins, CRP has become an important paradigm for allostery investigation. The crystal structures of CRP-cAMP, CRP-cAMP-DNA, and CRP-cAMP-DNA-RNAP complex have all been determined. The structure of CRP-cAMP complex revealed that one subunit is in a "closed" form, where the C-terminal domain swung in toward theα-helical interface, and the other subunit is in an "open" form, where the C-terminal domain swung away from theα-helical interface, and there is a large cleft between the two domains.However, the structure of CRP-cAMP-DNA complex showed that both subunits are in the "closed" form and are highly symmetric.The CRP has been studied extensively using methods ranging from biochemical, biophysical techniques and molecular dynamics,all show that the cAMP binding to CRP first provokes the local perturbation at the directly contacting points, which then propagates to whole molecule and leads to global transition. Recently, the structure thought to be the last one for the understanding of allostery mechanism induced by cAMP binding, apo-CRP, was determined.Although it was truly contributed to the understanding of the mechanism, there are many issues to be studied.A precisely determined structure of apo-CRP or the CRP-cAMP complex has a wider significance.The structure and allostery mechanism of cAMP ligated D138L mutant of CRP was studied in this work. With the application of biochemical,biophysical and spectrum technology, we got these conclusions:1.The D138L mutant of CRP was expressed,purified and crystallized.The structure was determined at a resolution of 1.66 A,which is by far the highest resolution structure of CRP. The coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3KCC.2.This high resolution structure of D138L-cAMP shows that there are four cAMPs binding to CRP, two are anti-cAMP, and the other two are syn-cAMP.3.Both subunits in D138L-cAMP complex are in the "closed" conformation, showing the highly symmetric.4.Comparison of the structure of liganded D138L CRP to the structure of unliganded D138L CRP reveals that there is very little conformational alteration in the N-terminal cAMP-binding domains except theβ-flap.On the other hand, the orientation of the DNA binding domains and the length of C and D helices differ dramatically between the two forms.5.Comparison of the closed subunit of wild type (WT) CRP with D138L CRP reveals that the most significant difference appears in theβ-flap which containsβ4,β5 and loop 3.These changes make the hinge region more flexible and sensitive to protease.6.Proteolytic digestions and Hydrogen-deuterium (H-D) exchanges by FT-IR spectroscope were employed to investigate protein dynamics.The results showed that the conformation of D138L mutant is much more dynamic than that of WT, which may impact the recognition of specific DNA sequence.Because of the cAMP-binding motifs founded in other proteins, the role of loops 3 and 4 in the interdomains and intersubunits communication of CRP were also studied.Two mutants were constructed according to the differences in sequence alignment between CRP and the other cAMP-dependent proteins, namely the insertion KGSKM between E78 and G79 in loop 4 (Mutl),and then the deletion of E54-G56 in loop 3 (Mut2).Together with the results of biochemical and biophysical experiments, we got these conclusions:1.To examine the structure integrity of WT, Mutl and Mut2,the overall structures at secondary and tertiary levels were determined by Near-UV CD,Far-UV CD and FTIR spectroscopes.The results show that there are no observable difference in the secondary and tertiary structures between the two mutants and the WT CRP.2.The fluorescence anisotropy experiment was used for quantitative analysis of CRP-DNA binding affinity in the presence of cAMP. The results show that the DNA binding affinity was perturbed by the insertion of KGSKM in the loop 4,while the deletion EEG in the loop 3 did not change the lac26 binding affinity. Thus,the loop 4 must play a role in the allosteric signal transmitting process, and involves in the interdomains communication.3.The isothermal titration calorimeter (ITC) was used to examine the cAMP binding affinity. The relationship between these differences and the structures were discussed.And the results showed that there are two strong cAMP-binding sites and a weaker cAMP-binding site for WT and the two mutants.The cAMP binding affinities for the Mutl CRP are much lower than that of WT, but did not change the cooperativity of the two binding sites, indicating that the loop 4 is not involved in intersubunits communication. There is a positive cooperativity between two strong binding sites of the Mut2 CRP, indicating that the loop 3 is involved in intersubunits communication.4.The changes in loop 3 and 4 perturb the cAMP binding pocket and alter cAMP binding behavior. The allosteric signal transmission process is most likely the result of overall realignment of the two subunits within a CRP dimer.5.Two models have been proposed to address the signal transmission pathway. The first model states that the conformational effect induced by cAMP is transmitted to the DNA binding site mainly through the hinge region in the monomer unit. The second model states that the overall realignment of the two subunits within CRP dimer is crucial for the activation of CRP upon cAMP binding.Our results support the latter.