Substrate-induced domain movement in a bifunctional protein, DcpA, regulates cyclic di-GMP turnover: Functional implications of a highly conserved motif

Abstract

In eubacteria, cyclic-di-GMP (c-di-GMP) signaling is involved in virulence, persistence, and motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). C-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multi-domain protein whose GAF-GGDEF-EAL domains are arranged in tandem and are required for these two activities. To gain insight into how interactions among these three domains affect DcpA activity, here we studied its domain dynamics using realtime FRET. We demonstrate that substrate binding in DcpA results in domain movement that prompts a switch from an "open" to a "closed" conformation and alters its catalytic activity. We found that a single point mutation in the conserved EAL motif (E384A) results in complete loss of the PDE activity of the EAL domain and in a significant decrease in the DGC activity of the GGDEF domain. Structural analyses revealed multiple hydrophobic and aromatic residues around Cys-579 that are necessary for proper DcpA folding and maintenance of the active conformation. On the basis of these observations and taking into account additional bioinformatics analysis of EAL domaincontaining proteins, we identified a critical putatively conserved motif, GCxxxQGF that plays an important role in c-di-GMP turnover. We conclude that a substrate-induced conformational switch involving movement of a loop containing a conserved motif in the bifunctional diguanylate cyclase-phosphodiesterase DcpA controls c-di-GMP turnover in M. smegmatis.

Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria

“…Binding of GDP to the GAF domain of DcpA enhances c-di-GMP hydrolysis and decreases synthesis (Chen et al 2018). An important aspect of its activity regulation comes from the substrate-induced domain movement (Bharati et al 2018). This was elegantly demonstrated by a Förster Resonance Energy Transfer (FRET)-based technique for studying relative domain movement (Fig.…”

Homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) fromMycobacterium smegmatis

“…In-gel tryptic digestion was performed to confirm the MsDisA, MsPDE proteins and their domain variants by a standard protocol with some modifications (65,66). The HPLC eluted fractions from the MsDisA and MsPDE reactions were analyzed by the LC-MS method (BurkerDaltonics, Germany) (57). MS-MS analysis (Negative or positive ion mode) of eluted molecular masses were performed to verify the presence of c-di-AMP and AMP.…”

“…M. smegmatis mc 2 155 and Escherichia coli strains used in this study are described in Table S1. M. smegmatis mc 2 155 strain was grown in MB7H9 medium (Difco) or MB7H9 medium solidified with 1.5% (w/v) agar with additional 2% glucose (vol/vol) and 0.05% Tween 80 (57). E. coli strains DH5α or BL21 (DE3) were grown in Luria-Bertani (LB) medium or in LB medium containing 1.5% (w/v) agar (58).…”

“…MsDisA (372 aa) and MsPDE (340 aa) proteins were purified using standard Ni-NTA column chromatography as described before (57,59). Briefly, E. coli BL21(DE3) cells containing plasmids for disA and pde were inoculated in LB medium (supplemented with 35 µg/ml of kanamycin), followed by their growth overnight at 37 0 C. Secondary cultures were prepared by inoculating 1% of the primary culture and grown at 37 0 C with shaking till the OD 600 reached ∼0.6.…”

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts

“…On the other hand, although in principle the hydrolysis of c-di-GMP could be performed by a single EAL domain, EAL containing proteins have been also shown to function as dimers and, as the GGDEF domains, to function as conformational switches [ 5 , 6 , 7 , 8 ]. Finally, the GGDEF domain can also be found fused at the N-terminus of an EAL domain, in the so-called hybrid proteins which potentially could display opposite catalytic activities.…”