Insights into Biofilm Dispersal Regulation from the Crystal Structure of the PAS-GGDEF-EAL Region of RbdA from Pseudomonas aeruginosa

Abstract

RbdA is a positive egulator ofiofilm ispersal of 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/Mg 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 (α) 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.Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of 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.

Insights into the GTP‐dependent allosteric control of c‐di‐GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonas aeruginosa

“…In agreement with this observations, the structural superposition of DUAL with the two homologues MorA [4] and RbdA [7] shows not only that the hinge helix is very flexible, but also that the GGDEF domain is able to assume any possible orientation with respect to the EAL domain (Fig. In agreement with this observations, the structural superposition of DUAL with the two homologues MorA [4] and RbdA [7] shows not only that the hinge helix is very flexible, but also that the GGDEF domain is able to assume any possible orientation with respect to the EAL domain (Fig.…”

“…In agreement with this observations, the structural superposition of DUAL with the two homologues MorA [4] and RbdA [7] shows not only that the hinge helix is very flexible, but also that the GGDEF domain is able to assume any possible orientation with respect to the EAL domain (Fig. Indeed a large conformational rearrangement upon GTP binding, leading to a more elongated active dimer, has also been proposed for RbdA [7]. Therefore, the two conformations observed in the asymmetric dimer of DUAL may represent two intermediate structural snapshots of the events following GTP binding and leading to a fully active ON species.…”

“…6A); regardless of metal content, the GTP binding mode is the same in both subunits and similar to that observed in RbdA [7]. Together with GTP, one calcium ion is octahedrally coordinated in monomer-B, while two calcium ions are present in monomer-A (Fig.…”

“…To this aim, we investigated the ability of monomer-A to reach a fully closed state, performing an all-atom normal mode (NM) analysis (Movies S1 and S2; [23,24]). Albeit it was not possible in the latter case to obtain a symmetric dimer of the OFF state, we expect that in such state the EAL domains would be 'locked' in a confirmation that prevents their dimerization, via close contacts involving residues of EAL and GGDEF domains, as observed in other GGDEF-EAL structures [7,25]. 8A,C), in which the a1-helix of the EAL domain is very close to the GTP binding site of the GGDEF domain.…”

“…Representative of this complexity are the so-called hybrid proteins, where the GGDEF and EAL domains are fused into the same polypeptide chain. In these proteins, the GGDEF-EAL pair may display a wide range of functional behaviours: the two domains may act as a genuine DGC and PDE, as in Pseudomonas aeruginosa MorA [4], or the GGDEF domain may regulate the PDE activity of the EAL domain, as in CC3396 from Caulobacter crescentus and in P. aeruginosa RbdA [5][6][7]. In these proteins, the GGDEF-EAL pair may display a wide range of functional behaviours: the two domains may act as a genuine DGC and PDE, as in Pseudomonas aeruginosa MorA [4], or the GGDEF domain may regulate the PDE activity of the EAL domain, as in CC3396 from Caulobacter crescentus and in P. aeruginosa RbdA [5][6][7].…”