Structural and Enzymatic Characterization of a cAMP-Dependent Diguanylate Cyclase from Pathogenic Leptospira Species. Issue 15 (21st July 2017)

• Record Type: Journal Article
• Authors: da Costa Vasconcelos, Fernanda Nogales; Maciel, Nikolas Koshiyama; Favaro, Denize Cristina; de Oliveira, Luciana Coutinho; Barbosa, Angela Silva; Salinas, Roberto Kopke; de Souza, Robson Francisco; Farah, Chuck Shaker; Guzzo, Cristiane Rodrigues
• Is Part Of: Journal of molecular biology. Volume 429:Issue 15(2017)
• Page Start: 2337
• Page End: 2352
• Publication Date: 2017-07-21
• Subjects: cAMP 3′, 5′-cyclic adenosine monophosphate -- cGMP 3′, 5′-cyclic guanosine monophosphate -- c-di-GMP bis-(3′→5′) cyclic dimeric guanosine monophosphate -- DGC diguanylate cyclase -- PDE phosphodiesterase -- GAF cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA -- cNMP cyclic mononucleotide -- SEC size-exclusion chromatography -- DOSY diffusion-ordered NMR spectroscopy -- CD circular dichroism -- CRP cAMP Receptor Protein; diguanylate cyclases -- bacterial signaling -- enzymatic kinetics -- X-ray crystallography -- c-di-GMP; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.06.002
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Leptospira interrogans serovar Copenhageni is a human pathogen that causes leptospirosis, a worldwide zoonosis. The L. interrogans genome codes for a wide array of potential diguanylate cyclase (DGC) enzymes with characteristic GGDEF domains capable of synthesizing the cyclic dinucleotide c-di-GMP, known to regulate transitions between different cellular behavioral states in bacteria. Among such enzymes, LIC13137 (Lcd1), which has an N-terminal c G MP-specific phosphodiesterases, a denylyl cyclases, and F hlA (GAF) domain and a C-terminal GGDEF domain, is notable for having close orthologs present only in pathogenic Leptospira species. Although the function and structure of GGDEF and GAF domains have been studied extensively separately, little is known about enzymes with the GAF-GGDEF architecture. In this report, we address the question of how the GAF domain regulates the DGC activity of Lcd1. The full-length Lcd1 and its GAF domain form dimers in solution. The GAF domain binds specifically cAMP ( K D of 0.24 μM) and has an important role in the regulation of the DGC activity of the GGDEF domain. Lcd1 DGC activity is negligible in the absence of cAMP and is significantly enhanced in its presence (specific activity of 0.13 s − 1 ). The crystal structure of the Lcd1 GAF domain in complex with cAMP provides valuable insights toward explaining its specificity for cAMP and pointing to possible mechanisms by which this cyclic nucleotide regulates the assembly of an active DGC enzyme. Graphical Abstract: Highlights: Lcd1 (LIC13137), a protein with GAF and GGDEF domains, is a DGC enzyme that synthesizes c-di-GMP. Lcd1 binds cAMP by the GAF domain, which enhances the DGC activity of the GGDEF domain. Lcd1 is a potential node for the integration of cAMP and c-di-GMP signaling in L. interrogans . GAF domain structure in complex with cAMP explains its specificity for cAMP ligand and suggests possible mechanisms for enzyme activation. Both full-length Lcd1 and its GAF domain form dimers in solution.