Oligomer Formation of the Bacterial Second Messenger c-di-GMP: Reaction Rates and Equilibrium Constants Indicate a Monomeric State at Physiological Concentrations

Abstract

Cyclic diguanosine-monophosphate (c-di-GMP) is a bacterial signaling molecule that triggers a switch from motile to sessile bacterial lifestyles. This mechanism is of considerable pharmaceutical interest, since it is related to bacterial virulence, biofilm formation, and persistence of infection. Previously, c-di-GMP has been reported to display a rich polymorphism of various oligomeric forms at millimolar concentrations, which differ in base stacking and G-quartet interactions. Here, we have analyzed the equilibrium and exchange kinetics between these various forms by NMR spectroscopy. We find that the association of the monomer into a dimeric form is in fast exchange (<milliseconds) with an equilibrium constant of about 1 mM. At concentrations above 100 μM, higher oligomers are formed in the presence of cations. These are presumably tetramers and octamers, with octamers dominating above about 0.5 mM. Thus, at the low micromolar concentrations of the cellular environment and in the absence of additional compounds that stabilize oligomers, c-di-GMP should be predominantly monomeric. This finding has important implications for the understanding of c-di-GMP recognition by protein receptors. In contrast to the monomer/dimer exchange, formation and dissociation of higher oligomers occurs on a time scale of several hours to days. The time course can be described quantitatively by a simple kinetic model where tetramers are intermediates of octamer formation. The extremely slow oligomer dissociation may generate severe artifacts in biological experiments when c-di-GMP is diluted from concentrated stock solution. We present a simple method to quantify c-di-GMP monomers and oligomers from UV spectra and a procedure to dissolve the unwanted oligomers by an annealing step.

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

“…It has been reported that c-di-GMP forms heterogeneous oligomers (including monomer, dimer, tetramer, and octamer) in solution (36). Because only the monomer of c-di-NMP can enter the active site, we further tested whether the difference in , and the bottom line shows that 5Ј-pApA is released first and then binds back to Rv2837c.…”

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

“…After removing the volatile buffer and the eluent by freeze-drying, the powder was dissolved in water. The concentration of the nucleotide was calculated by recording absorbance at 253 nm and using an absorption coefficient of 28Ј590 M Ϫ1 cm Ϫ1 (18). The purity of the product was checked by mass spectrometry.…”

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…Both forms were subsequently found in crystal structures of c-di-GMP-binding and -metabolizing proteins (36, 63-65, 75, 82-86). Cyclic di-GMP can also form higher oligomers, tetramers, and even octamers (87); their physiological roles, if any, remain unknown. Shortly after discovering c-di-GMP, Benziman's group identified and sequenced the genes encoding enzymes responsible for its synthesis and breakdown, i.e., the diguanylate cyclase (DGC) and c-di-GMP-specific phosphodiesterase (PDE), respectively.…”