Structural And Functional Studies On CAMP Receptor Protein From Gardnerella Vaginalis

Bacterial vaginosis(BV)is the most common vaginal disease,mainly caused by excessive growth of Gardnerella vaginalis(GV).BV will cause itching,burning,pain,sretion odor and other symptoms,and increase the risk of sexually transmitted disease infection,pelvic inflammation,infertility and preterm birth.The global prevalence of BV is as high as 29%,and the main treatment remains antibiotics.Nearly 9.6 billion dollars are spent annually for curing BV.The pathogenicity of GV is mainly in the formation of biofilms(enhancing its resistance)and the release of virulence factors(killing host cells).Formation of biofilms and transcription of virulence factors are directly or indirectly regulated by the cyclic adenosine monophosphate receptor protein(CRP).The conformational change of the CRP csused by binding to the ligand affects the KD to promoter DNA.The drugs designed for the CRP structure of the pathogenic bacteria could be a new treatment of BV,so it is of great significance to explore the CRP structure and allosteric mechanism,which help find potential drug targets.This thesis mainly obtained the following research results:1.Determine the structure of CRPGv at 2.22 (?)CRPGv with more than 95%purity was obtained by expression and purification.After the general screening and optimization steps,good-quality crystals were obtained.Diffraction data were collected and the structure was determined at 2.22 (?).The structure showed that:the main structure of CRPGv was similar to its homologous protein,but the first twenty-three amino acid of CRPGv was inserted into the cAMP binding domain cavity,which was in the different state with most other homologous proteins;the two helixes binding DNA were exposed,and the distance was 35.1(?),less than the homologous protein,which indicated CRPGv may have DNA binding ability;the structure contained no small molecule ligands.2.The ligand of CRPGv was not known ligandsFrom the bioinformatics analysis,cAMP is most likely CRPGv’s ligand.However,after multiple identification,such as ITC,VP-DSC,HPLC,EMSA,CD,and trypsin tolerance experiments,cAMP failed to interact with CRPGv.Post-experiment with cGMP,c-di-GMP,cdi-AMP,OCPA,α-Ketoglutaric acid,showed no effect on CRPGv,and was not CRPGv’s allosteric ligand.This suggested that there may be new regulation form in CRPGv(allosteric regulation with unknown ligands,or with interacting proteins).3.CRPGv binds to the DNA box of TGTGA-N6-TCACAThe promoter DNA sequences of GV were predicted using the promoter prediction website.Then using EMSA experiments to find target promoter,Promoter 29 exhibited strong affinity.Its sequence was only one nucleotide different from the CRP classical box sequence TGTGA-N6-TCACA,and EMSA analysis with the box sequence showed stronger affinity,indicating that TGTGA-N6-TCACA was CRPGv’s binding box.CRPGv in an active conformation provided a structural basis for screening drugs.4.Amino acid mutations at the key site had no effect on CRPGvUsing CRP mutants could be an affective way to explore the allosteric mechanism of CRPGv.Corresponding to mutations published in previous studies,CRPGv’s mutants were constructed,including A162D,A162S,D159L,D159N,K166S,N148L/N149I,N149I,R163H,N148L,and these protein’s structure were predicted using AlphaFold 2.0.After experiment,these mutant sites causing homologous proteins for allosterism and activity change were not applicable to CRPGv and produced no changes in structure and function.