π-Helix controls activity of oxygen-sensing diguanylate cyclases.

Johnnie A Walker,Yuqi Wu,Emily E Weinert,Jacob R Potter

doi:10.1042/bsr20193602

Johnnie A Walker, Yuqi Wu + Show 2 more

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https://doi.org/10.1042/bsr20193602

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Abstract

The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

Highlights

Since the discovery of cyclic dimeric guanosine monophosphate (c-di-GMP) as a bacterial second messenger and its effects on biofilm formation and antibiotic resistance, the proteins involved in c-di-GMP pathways have been of considerable interest as possible targets for the development of new antibiotics [1–4]
Structures have been solved for a handful of full-length regulatory domain-diguanylate cyclase (DGC) proteins and show two general domain organizations: (1) regulatory domain separated from DGC domain by an extended linker and (2) regulatory domain and DGC domain in direct contact due to a very short/absent linking domain [16,33,34]
Activation of the regulatory domain in the second case results in rotation of the DGC domains to align the active site and allow for c-di-GMP production

Summary

Introduction

Since the discovery of cyclic dimeric guanosine monophosphate (c-di-GMP) as a bacterial second messenger and its effects on biofilm formation and antibiotic resistance, the proteins involved in c-di-GMP pathways have been of considerable interest as possible targets for the development of new antibiotics [1–4]. To date, conserved motifs for the enzymes responsible for c-di-GMP production (diguanylate cyclase; GGDEF) and c-di-GMP hydrolysis (c-di-GMP phosphodiesterase; EAL or HDGYP) have been identified, allowing for relatively facile identification of bacteria that utilize c-di-GMP signaling to control cellular phenotypes [5,6]. Our understanding of how c-di-GMP metabolic enzymes are regulated and how bacteria obtain disparate downstream effects from activation of different diguanylate cyclases is incomplete. Oxygen (O2) levels have been demonstrated to regulate biofilm formation and to affect virulence, making it an important external signal, likely due to its importance in metabolism [9–11]. Globin coupled sensor (GCS) proteins utilize a heme-containing globin domain to sense O2 levels and transmit the ligand-binding signal through a linking middle domain to an output domain.

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