Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation

The COMBREX Project: Design, Methodology, and Initial Results

© 2013 Brian P. et al.Prior to the “genomic era,” when the acquisition of DNA sequence involved significant labor and expense, the sequencing of genes was strongly linked to the experimental characterization of their products. Sequencing at that time directly resulted from the need to understand an experimentally determined phenotype or biochemical activity. Now that DNA sequencing has become orders of magnitude faster and less expensive, focus has shifted to sequencing entire genomes. Since biochemistry and genetics have not, by and large, enjoyed the same improvement of scale, public sequence repositories now predominantly contain putative protein sequences for which there is no direct experimental evidence of function. Computational approaches attempt to leverage evidence associated with the ever-smaller fraction of experimentally analyzed proteins to predict function for these putative proteins. Maximizing our understanding of function over the universe of proteins in toto requires not only robust computational methods of inference but also a judicious allocation of experimental resources, focusing on proteins whose experimental characterization will maximize the number and accuracy of follow-on predictions.COMBREX is funded by a GO grant from the National Institute of General Medical Sciences (NIGMS) (1RC2GM092602-01).Peer Reviewe

Structure and regulation of EAL domain proteins

The formation and dispersal of bacterial biofilms is strongly correlated with cellular levels of bis-(3′–5′) cyclic dimeric guanosine monophosphate, cyclic di-GMP, a secondary messenger that has been shown to be involved in regulation of a broad range of cellular processes in bacteria. Diguanylate cyclases (DGCs) are required for synthesis of cyclic di-GMP, with phosphodiesterases (PDEs) responsible for its breakdown. This review focuses on PDEs characterised by the presence of the conserved “EAL” sequence motif. Typically found in multi-domain proteins, EAL domains can couple to sensory or regulatory domains that allow their activity to be regulated by environmental stimuli or cellular cues. Additionally, catalytically inactive EAL PDEs are suggested to have a sensory or otherwise regulatory function. Recent structure determination provides a wealth of information on PDE function and regulation and has provided novel insight into the enzymatic reaction mechanism. Several regulatory layers may control activity, including dimerisation, active site formation, and metal coordination. In this review, we provide a concise summary of these exciting findings and highlight open research questions that will allow us in future to decipher many of the cellular signals responsible for regulation of PDE activity and cellular processes influenced by these pivotal enzymes