Expression, Purification and Characterization of c-di-GMP Binding Protein YcgR from Salmonella typhimurium

Abstract:

[Background] In the Salmonella typhimurium, the c-di-GMP binding protein YcgR could inhibit flagellar motor speed by interacting with flagellar protein, which cause the bacteria stop migrate, generate biofilm and infect the host. However, the structure and functions of YcgR as well as the molecular mechanism of YcgR suppressing bacterial motion after binding to c-di-GMP are not clear. [Objective] In this study, YcgR protein was expressed and purified in recombinant E. coli, and then the c-di-GMP binding activity of YcgR were further characterized, which could lay a foundation for determining the structure of YcgR and clarifying how c-di-GMP binding protein YcgR slowing the movement of Salmonella typhimurium. [Methods] The recombinant E. coli was constructed for expressing YcgR protein, which was further purified by Ni-NTA and SEC (Size exclusion chromatography). After that, we predicted the structure of YcgR protein by comparing with other homologous family proteins, and the secondary structure of YcgR was determined by circular dichroism (CD). Finally, the binding parameters of YcgR and c-di-GMP were determined by isothermal titration calorimetry (ITC) and Protein Thermal Shift. [Results] Colony PCR and DNA sequencing results showed that the recombinant E. coli for expressing YcgR was successfully constructed. The size exclusion chromatography and polyacrylamide gel electrophoresis showed that the molecular weight of YcgR was 28 kD and existed as dimer in the supernatant. The structure of YcgR is highly similar to the homologous protein PP4396, which has a PilZ domain for binding c-di-GMP, and CD results showed that YcgR had a stable secondary structure. In addition, ITC and Thermal Shift indicated that YcgR had a strong and specific binding activity with c-di-GMP, and the Kd value was 50 nmol/L. [Conclusion] The YcgR protein from Salmonella typhimurium was successfully expressed in E. coli for the first time, and its c-di-GMP binding activity were also characterized in this study, which is very important for the study of the molecular mechanism of c-di-GMP binding protein YcgR regulating flagellin, and provide a new strategy for bacterial anti-infective therapy.