Structures of the catalytic EAL domain of the<i>Escherichia coli</i>direct oxygen sensor

Tarnawski, Mirosław; Barends, Thomas R. M.; Hartmann, Elisabeth; Schlichting, Ilme

doi:10.1107/s0907444913004423

Cited by 9 publications

(7 citation statements)

References 37 publications

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“…Structural alignments with respect to protomers A of the characteristic EAL dimers of YkuI (UNP ID: O35014) [24], BlrP1 [10], Td EAL (UNP ID: Q3SJE6) [13], Cc EAL (UNP ID: Q9A310 and PDB ID: 3U2E; unpublished), RocR (UNP ID: Q9HX69) [40] and DosP (UNP ID: P76129) [39] are shown in cartoon representation. The orientations of the upper two panels correspond to those of Fig.…”

Section: Figmentioning

confidence: 99%

Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

Winkler¹,

Udvarhelyi²,

Hartmann³

et al. 2014

Journal of Molecular Biology

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Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen–deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen–deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.

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Section: Figmentioning

confidence: 99%

Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

Winkler¹,

Udvarhelyi²,

Hartmann³

et al. 2014

Journal of Molecular Biology

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“…For EcDOS, a scissor-like movement of the subunits in the isolated PAS dimer upon heme cofactor reduction has been reported (34). This motion may be propagated to the EAL domain that has been observed to exist as a canonical dimer in isolation (32).…”

Section: Table 2 Ligand Dissociation Constants (Nm)mentioning

confidence: 99%

“…32 and references therein), the substrate is bound to an extended shallow groove at the C-terminal end of the ␤-barrel (Fig. 4B).…”

Section: Structural Basis-highmentioning

confidence: 99%

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

Sundriyal

Massa

Samoray

et al. 2014

Journal of Biological Chemistry

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Background:The bacterial second messenger cyclic di-GMP (c-di-GMP) is degraded by EAL phosphodiesterases. Results:The isolated EAL domain is active only as a homodimer. Substrate binding is coupled with EAL dimerization. Conclusion: Activity of many full-length EAL phosphodiesterases may be regulated by catalytic domain dimerization. Significance: A generic mechanism for the regulation of a central node of c-di-GMP signaling is provided.

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“…A detailed mechanism for the hydrolysis of c‐di‐GMP by PDEs has not been worked out, but analyses of crystal structures of PDEs with or without bound ligands have provided some clues regarding how c‐di‐GMP is cleaved. A survey of the known crystal structures of proteins with the EAL domain PDEs is presented in the article by Tarnawski and co‐authors; we mention here only those relevant to our study.…”

Section: Introductionmentioning

confidence: 99%

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

2016

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We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc.

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Structures of the catalytic EAL domain of theEscherichia colidirect oxygen sensor

Cited by 9 publications

References 37 publications

Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

Characterization of Elements Involved in Allosteric Light Regulation of Phosphodiesterase Activity by Comparison of Different Functional BlrP1 States

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

Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

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