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

Bharati, Binod K and Mukherjee, Raju and Chatterji, Dipankar (2018) Substrate-induced domain movement in a bifunctional protein, DcpA, regulates cyclic di-GMP turnover: Functional implications of a highly conserved motif. In: JOURNAL OF BIOLOGICAL CHEMISTRY, 293 (36). pp. 14065-14079.

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Official URL: http://dx.doi.org/10.1074/jbc.RA118.003917


In eubacteria, cyclic di-GMP (c-di-GMP) signaling is involved in virulence, persistence, 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). The c-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multidomain 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 real-time 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 domain-containing 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.

Item Type: Journal Article
Additional Information: Copy right for this article belong to AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USA
Keywords: cyclic di-GMP (c-di-GMP); fluorescence resonance energy transfer (FRET); c-di-GMP turnover; DcpA; diguanylate cyclase; phosphodiesterase; domain movement; Forster's distance; second messenger; bacterial virulence; fluorescence anisotropy
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Division of Interdisciplinary Sciences > Supercomputer Education & Research Centre
Date Deposited: 26 Sep 2018 15:27
Last Modified: 26 Sep 2018 15:27
URI: http://eprints.iisc.ac.in/id/eprint/60732

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