Crystal structure reveals mechanism of biofilm dispersal
Research from SCELSE has identified important features of the RbdA protein that can be used to target Pseudomonas biofilm.
- Biofilm dispersal in Psuedomonas aeruginosa is regulated by RbdA
- The structure of the cytoplasmic portion reveals 2 regions that could be used to find small molecules to promote biofilm dispersion
- Structural similarities with other bacterial signalling proteins could give functional insights across a wide selection of bacteria
Researchers from NTU and SCELSE have been addressing biofilm formation at the atomic level. The team led by Julien Lescar and Scott Rice focussed on the Ps. aeruginosa protein RbdA which is known to regulate biofilm dispersal. The cytoplasmic portion of the RbdA (cRbdA) was crystallised and and diffraction patterns were measured at two different synchrotron facilities, one in Australia (Australian Synchrotron) and one in Switzerland (Swiss Light Source) which resulted in a crystal structure. The structural information allowed the team to hypothesise a switching mechanism whereby the conformation of the protein changes and "unlocks" depending on the binding of GTP. This mechanism was confirmed using small angle X-ray Scattering or SAXS. Identifying the structure and the control mechanism represents an important breakthrough in understanding biofilm formation in Ps. aeruginosa and paves the way to find molecular inhibitors or promotors of biofilm dispersal. The similarity of the structure to other signalling proteins found in bacteria may help to discern previously unknown modes of action.
RbdA is a positive Regulator of biofilm dispersal of Pseudomonas aeruginosa Its cytoplasmic region (cRbdA) comprises a N-terminal PAS domain followed by a diguanylate cyclase (GGDEF) and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: with GTP/Mg2+ bound to the GGDEF active site and with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer, stabilized by a closely packed N-terminal PAS domain and a non-canonical EAL dimer. The auto-inhibitory switch is formed by an alpha helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (α6-E) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by SAXS experiments. Domain reorientation should be facilitated by the presence of a α-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.IMPORTANCEBiofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of Pseudomonas aeruginosa The crystal structures of the cytoplasmic region of RbdA protein presented here reveal that two evolutionary-conserved helices play an important role in regulating the activity of RbdA, with implications for other dual GGDEF-EAL domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work could assist the development of small molecules that would promote bacterial biofilm dispersal.
J Bacteriol. 2017 Nov 6. pii: JB.00515-17. doi: 10.1128/JB.00515-17. [Epub ahead of print]
Insights into biofilm dispersal regulation from the crystal structure of the PAS-GGDEF-EAL region of RbdA from Pseudomonas aeruginosa.
Liu C, Liew CW, Wong YH, Tan ST, Poh WH, Manimekalai SMS, Rajan S, Xin L, Liang ZX, Grüber G, Rice SA, Lescar J4.