Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

Abstract

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

Heme-Edge Residues Modulate Signal Transduction within a Bifunctional Homo-Dimeric Sensor Protein

“…To gain insights into the conformational changes that could be linked to the mutations, SAXS and negative stain EM were used to probe the heme-edge variants and compared to WT models (Figure A,B) . The DcpG WT was found to form a tight globin dimer with EAL active sites dimerized for high activity.…”

“…5′-Nucleotidase from Crotalus atrox venom (Enzo Life Sciences) also was added to the kit at a concentration of 100 units/mL per well. 5′-Nucleotidase catalyzes the hydrolysis of pGpG to GpG and phosphate but does not hydrolyze c-di-GMP . A360 readings were monitored every 30 s for 180 min .…”

“…5′-Nucleotidase catalyzes the hydrolysis of pGpG to GpG and phosphate but does not hydrolyze c-di-GMP . A360 readings were monitored every 30 s for 180 min . A360 readings were monitored every 30 s for 180 min.…”

“…GCS proteins consist of an N-terminal sensor globin domain linked by a middle domain of varying length to an output domain. In the case of DcpG, the protein contains both c-di-GMP synthesis (GGDEF) and hydrolysis (EAL) domains that are differentially regulated by O 2 and NO . C-di-GMP is a bacterial second messenger that regulates biofilm formation and is an important regulator of motility and virulence .…”

“…In contrast, O 2 binding results in activation of c-di-GMP hydrolysis by the EAL domain, relative to Fe II unligated, while NO (and CO) have no significant effects. Using small-angle x-ray scattering (SAXS) and negative stain electron microscopy (EM), we demonstrated that DcpG in the Fe­(II)-O 2 ligation state forms a dimer that physically distances the GGDEF domains, inhibiting activity, while allowing formation of the active EAL domain dimer . In addition, DcpG was found to exhibit surprisingly rapid O 2 and NO dissociation rates from the sensor globin domain and, using spectro-electrochemistry, the globin domain was demonstrated to have two distinct high midpoint potentials (+418 and 279 mV vs the standard hydrogen electrode (SHE)), demonstrating the non-equivalency of the hemes within the DcpG homodimer …”